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Sample records for bcl2-derived epitope expressed

  1. Identification of an HLA-A*0201 restricted Bcl2-derived epitope expressed on tumors

    DEFF Research Database (Denmark)

    Wang, Mingjun; Johansen, Britta; Nissen, Mogens H

    2006-01-01

    A large number of human tumor-associated antigen-derived peptides have been identified that are recognized by CTLs in a MHC-I restricted fashion. The apoptosis inhibitory protein Bcl2 is overexpressed in many human cancers as part of their neoplastic phenotype. Since inhibition or loss of Bcl2...... expression might impair tumor growth and survival, this protein may serve as a rational target for vaccine-induced CTL responses. By Western blot technique, we screened a panel of established human tumor cell lines for proteins involved in the apoptotic process. Two of eight tumor cell lines, a B lymphoma...... (Loukes) and a colon carcinoma (CCL220) cell line showed increased Bcl2 protein expression whereas the majority of tumor cell lines expressed proapoptotic proteins. Neither fibroblasts nor peripheral blood mononuclear cells showed Bcl2 expression. An HLA-A*0201 restricted CTL epitope was deduced in silica...

  2. Expression and immunoreactivity of HCV/HBV epitopes

    Institute of Scientific and Technical Information of China (English)

    Xin-Yu Xiong; Xiao Liu; Yuan-Ding Chen

    2005-01-01

    AIM: To develop the epitope-based vaccines to prevent Hepatitis C virus (HCV)/Hepatitis B virus (HBV) infections.METHODS: The HCV core epitopes C1 STNPKPQRKTKRNTNRRPQD (residuals aa2-21) and C2 VKFPGGGQIVGGVYLLPRR (residuals aa22-40), envelope epitope E GHRMAWDMMMNWSP (residuals aa315-328) and HBsAg epitope S CTTPAQGNSMFPSCCCTKPTDGNC (residuals aa124-147) were displayed in five different sites of the flock house virus capsid protein as a vector, and expressed in E. coli cells (pET-3 system).Immunoreactivity of the epitopes with anti-HCV and anti-HBV antibodies in the serum from hepatitis C and hepatitis B patients were determined.RESULTS: The expressed chimeric protein carrying the HCV epitopes C1, C2, E (two times), L3C1-I2E-L1C2-L2E could react with anti-HCV antibodies. The expressed chimeric protein carrying the HBV epitopes S, I3S could react with anti-HBs antibodies. The expressed chimeric proteins carrying the HCV epitopes C1, C2, E plus HBV epitope S, L3C1-I2E-L1C2-L2E-I3S could react with antiHCV and anti-HBs antibodies.CONCLUSION: These epitopes have highly specific and sensitive immunoreaction and are useful in the development of epitope-based vaccines.

  3. Kinetics of antigen expression and epitope presentation during virus infection.

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    Nathan P Croft

    2013-01-01

    Full Text Available Current knowledge about the dynamics of antigen presentation to T cells during viral infection is very poor despite being of fundamental importance to our understanding of anti-viral immunity. Here we use an advanced mass spectrometry method to simultaneously quantify the presentation of eight vaccinia virus peptide-MHC complexes (epitopes on infected cells and the amounts of their source antigens at multiple times after infection. The results show a startling 1000-fold range in abundance as well as strikingly different kinetics across the epitopes monitored. The tight correlation between onset of protein expression and epitope display for most antigens provides the strongest support to date that antigen presentation is largely linked to translation and not later degradation of antigens. Finally, we show a complete disconnect between the epitope abundance and immunodominance hierarchy of these eight epitopes. This study highlights the complexity of viral antigen presentation by the host and demonstrates the weakness of simple models that assume total protein levels are directly linked to epitope presentation and immunogenicity.

  4. Expression of goose parvovirus whole VP3 protein and its epitopes in Escherichia coli cells.

    Science.gov (United States)

    Tarasiuk, K; Woźniakowski, G; Holec-Gąsior, L

    2015-01-01

    The aim of this study was the expression of goose parvovirus capsid protein (VP3) and its epitopes in Escherichia coli cells. Expression of the whole VP3 protein provided an insufficient amount of protein. In contrast, the expression of two VP3 epitopes (VP3ep4, VP3ep6) in E. coli, resulted in very high expression levels. This may suggest that smaller parts of the GPV antigenic determinants are more efficiently expressed than the complete VP3 gene.

  5. Mapping of B-Cell Epitopes in a Trypanosoma cruzi Immunodominant Antigen Expressed in Natural Infections

    Science.gov (United States)

    Lesénéchal, Mylène; Becquart, Laurence; Lacoux, Xavier; Ladavière, Laurent; Baida, Renata C. P.; Paranhos-Baccalà, Glaucia; da Silveira, José Franco

    2005-01-01

    Tc40 is an immunodominant antigen present in natural Trypanosoma cruzi infections. This immunogen was thoroughly mapped by using overlapping amino acid sequences identified by gene cloning and chemical peptide synthesis. To map continuous epitopes of the Tc40 antigen, an epitope expression library was constructed and screened with sera from human chagasic patients. A major, linear B-cell epitope spanning residues 403 to 426 (PAKAAAPPAA) was identified in the central domain of Tc40. A synthetic peptide spanning this region reacted strongly with 89.8% of the serum samples from T. cruzi-infected individuals. This indicates that the main antigenic site is defined by the linear sequence of the peptide rather than a conformation-dependent structure. The major B-cell epitope of Tc40 shares a high degree of sequence identity with T. cruzi ribosomal and RNA binding proteins, suggesting the existence of cross-reactivity among these molecules. PMID:15699429

  6. [Construction of fusion gene vaccine of WT1 multi-epitope fused with stimulating epitope of mycobacterium tuberculosis heat shock protein 70 and its expression and immunogenicity].

    Science.gov (United States)

    Tian, Wei-Wei; Qiao, Zhen-Hua; Yang, Lin-Hua; Wang, Hong-Wei; Tang, Yan-Hong; Bian, Si-Cheng

    2011-04-01

    This study was purposed to construct a fusion DNA vaccine containing WT1 multi-epitope and stimulating epitope of mycobacterium tuberculosis heat shock protein 70 and to detect its expression and immunogenicity. On the basis of published data, a multi-epitope gene (Multi-WT1) containing three HLA *0201-restricted CTL epitopes: one HLA*2402-restricted CTL epitope, two Th epitopes and one universal Th Pan-DR epitope (PADRE) was constructed. DNA-coding sequence was modified by Computer-Aided Design (CAD) to optimize proteasome-mediated epitope processing through the introduction of different amino acid spacer sequences. The synthetic nucleotide sequence was then inserted into an eukaryotic vector to construct the plasmid pcDNA3.1-WT1.For enhancing CTL activity, HSP70 fragment including stimulatory domain P407-426 was amplified by PCR from mycobacterial HSP70 gene and cloned into pcDNA3.1(+). Then Multi-WT1 was fused to the N-terminal of pcDNA3.1-mHSP70(407-426) to make the multi-epitope fusion gene vaccine pcDNA3.1-WT1-mHSP70(407-426). HEK-293T cells were transfected with this vaccine and the expressed product was identified by RT-PCR. Enzyme-linked immunospot assay (ELISPOT) was used to evaluate the immunological responses elicited by vaccine. The results showed that the most of WT1 epitopes could be correctly cleaved which was confirmed by software Net Chop 3.1 and PAPROCIanalysis. RT-PCR showed correct expression of target gene in HEK293T cells and ELISPOT showed specific T-cell responses. It is concluded that the eukaryotic expression vector PcDNA3.1-WT1-mHSP70(407-426) fusion gene has been successfully constructed and the immunity response is also elicited, which is a good candidate for further research of DNA vaccine.

  7. Epitope specificity and V gene expression of cerebrospinal fluid T cells specific for intact versus cryptic epitopes of myelin basic protein.

    Science.gov (United States)

    Satyanarayana, K; Chou, Y K; Bourdette, D; Whitham, R; Hashim, G A; Offner, H; Vandenbark, A A

    1993-04-01

    Recent evidence supports the possible involvement of myelin basic protein (BP) as one of the target autoantigens in multiple sclerosis (MS), including elevated frequencies of MS blood and cerebrospinal fluid (CSF) T cells, and the presence in MS plaque tissue of V beta gene sequences and CDR3 motifs characteristic of BP-reactive T cells. Because of its proximity to the target organ, the CSF has long been thought to harbor T cells involved in the pathogenic process. In order to evaluate their frequency and response characteristics, BP-reactive T cells were isolated by limiting dilution from the CSF of patients with MS and other neurological diseases (OND) for quantitation and determination of epitope specificity and V alpha and V beta gene expression. In addition to isolates responsive to intact BP epitopes that were present at a significantly higher frequency in MS versus OND CSF, we here describe a second clonotype responsive to 'cryptic' BP epitopes that is present at approximately equal frequencies in MS and OND patients. In spite of their difference in recognition of intact versus 'cryptic' BP determinants, both clonotypes predominantly recognized epitopes in the N terminal half of human BP, using a similar V gene repertoire that included biased use of V alpha 2 and to a lesser degree V beta 7 and V beta 18. These V gene biases were not related to the epitope specificity of the T cells, indicating that V gene selection is not epitope-driven. These data suggest that there is differential recognition of intact versus 'cryptic' BP determinants in MS versus OND patients that may be related to the processing and presentation of BP to the immune system.

  8. Identification of swine influenza virus epitopes and analysis of multiple specificities expressed by cytotoxic T cell subsets

    DEFF Research Database (Denmark)

    Pedersen, Lasse Eggers; Breum, Solvej Østergaard; Riber, Ulla;

    2014-01-01

    to elimination of viruses such as swine influenza virus (SwIV). This study describes the identification of SLA-presented peptide epitopes that are targets for a swine CTL response, and further analyses multiple specificities expressed by SwIV activated CTL subsets. Findings: Four SwIV derived peptides were...... identified as T cell epitopes using fluorescent influenza: SLA tetramers. In addition, multiple CTL specificities were analyzed using peptide sequence substitutions in two of the four epitope candidates analyzed. Interestingly both conserved and substituted peptides were found to stain the CD4-CD8+ T cell...

  9. Tumor-associated antigens identified by mRNA expression profiling as tumor rejection epitopes

    DEFF Research Database (Denmark)

    Andersen, Marie Louise; Ruhwald, Morten; Thorn, Mette;

    2003-01-01

    immunization, but only two of these peptides (RAD23-31 and RAD24-31) were capable of generating a weak vaccination-induced protection against adoptive tumor growth. SM7 inoculated mice treated with a blocking antibody against the inhibitory T cell signal transducing molecule CTLA4 appeared to delay tumor take...... derived from potentially overexpressed tumor proteins, as identified by mRNA expression profiling of p53-/- thymoma cells, at best results in a weak tumor protection thus questioning this way of detection of new tumor rejection epitopes....

  10. Expressing Redundancy among Linear-Epitope Sequence Data Based on Residue-Level Physicochemical Similarity in the Context of Antigenic Cross-Reaction

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    Salvador Eugenio C. Caoili

    2016-01-01

    Full Text Available Epitope-based design of vaccines, immunotherapeutics, and immunodiagnostics is complicated by structural changes that radically alter immunological outcomes. This is obscured by expressing redundancy among linear-epitope data as fractional sequence-alignment identity, which fails to account for potentially drastic loss of binding affinity due to single-residue substitutions even where these might be considered conservative in the context of classical sequence analysis. From the perspective of immune function based on molecular recognition of epitopes, functional redundancy of epitope data (FRED thus may be defined in a biologically more meaningful way based on residue-level physicochemical similarity in the context of antigenic cross-reaction, with functional similarity between epitopes expressed as the Shannon information entropy for differential epitope binding. Such similarity may be estimated in terms of structural differences between an immunogen epitope and an antigen epitope with reference to an idealized binding site of high complementarity to the immunogen epitope, by analogy between protein folding and ligand-receptor binding; but this underestimates potential for cross-reactivity, suggesting that epitope-binding site complementarity is typically suboptimal as regards immunologic specificity. The apparently suboptimal complementarity may reflect a tradeoff to attain optimal immune function that favors generation of immune-system components each having potential for cross-reactivity with a variety of epitopes.

  11. Expression and stability of foreign epitopes introduced into 3A nonstructural protein of foot-and-mouth disease virus.

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    Pinghua Li

    Full Text Available Foot-and-mouth disease virus (FMDV is an aphthovirus that belongs to the Picornaviridae family and causes one of the most important animal diseases worldwide. The capacity of other picornaviruses to express foreign antigens has been extensively reported, however, little is known about FMDV. To explore the potential of FMDV as a viral vector, an 11-amino-acid (aa HSV epitope and an 8 aa FLAG epitope were introduced into the C-terminal different regions of 3A protein of FMDV full-length infectious cDNA clone. Recombinant viruses expressing the HSV or FLAG epitope were successfully rescued after transfection of both modified constructs. Immunofluorescence assay, Western blot and sequence analysis showed that the recombinant viruses stably maintained the foreign epitopes even after 11 serial passages in BHK-21 cells. The 3A-tagged viruses shared similar plaque phenotypes and replication kinetics to those of the parental virus. In addition, mice experimentally infected with the epitope-tagged viruses could induce tag-specific antibodies. Our results demonstrate that FMDV can be used effectively as a viral vector for the delivery of foreign tags.

  12. Intracellular processing and presentation of T cell epitopes, expressed by recombinant Escherichia coli and Salmonella typhimurium, to human T cells

    NARCIS (Netherlands)

    G.M.G.M. Verjans (George); C.M. Janssen (Riny); F.G.C.M. Uytdehaag (Fons); C.E.M. van Doornik (C. E M); J. Tommassen (Jan)

    1995-01-01

    textabstractVaccines based on recombinant attenuated bacteria represent a potentially safe and effective immunization strategy. A carrier system was developed to analyze in vitro whether foreign T cell epitopes, inserted in the outer membrane protein PhoE of Escherichia coli and expressed by recombi

  13. Repeated epitope in the recombinant epitope-peptide could enhance ELDKWA-epitope-specific antibody response

    Institute of Scientific and Technical Information of China (English)

    LIU Zuqiang; WANG Zuguang; CHEN Yinghua

    2005-01-01

    Based on the hypothesis suggested by us that epitope-vaccine may be a new strategy against HIV mutation, we have studied several neutralizing epitopes on HIV envelope proteins. However we do not know whether a repeated epitope in a recombinant epitope-peptide can enhance epitope-specific antibody response or not. ELDKWA-epitope (aa669-674) on the C-domain of HIV-1 gp41 is a neutralizing epitope defined by the monoclonal antibody (mAb) 2F5 with broad neutralizing activity. In this study, we designed and prepared a series of the recombinant epitope-peptides bearing 1, 4 and 8 copies of ELDKWA-epitope respectively. In the comparison of the antisera induced by the three recombinant antigens, an obviously increased titre of ELDKWA-epitope-specific antibody was observed in the case of four and eight repeated epitopes. In flow cytometry analysis, the epitope-specific antibodies in both antisera showed stronger activity to bind the transfected CHO-WT cells that stably express HIV-1 envelope glycoprotein on the cell surfaces. These experimental results indicated that repeated epitope in the recombinant epitope-peptide could enhance ELDKWA-epitope-specific antibody response, which could contribute to designing an effective recombinant epitope-vaccine.

  14. Expression and purification of chimeric peptide comprising EGFR B-cell epitope and measles virus fusion protein T-cell epitope in Escherichia coli.

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    Wu, Meizhi; Zhao, Lin; Zhu, Lei; Chen, Zhange; Li, Huangjin

    2013-03-01

    Chimeric peptide MVF-EGFR(237-267), comprising a B-cell epitope from the dimerization interface of human epidermal growth factor receptor (EGFR) and a promiscuous T-cell epitope from measles virus fusion protein (MVF), is a promising candidate antigen peptide for therapeutic vaccine. To establish a high-efficiency preparation process of this small peptide, the coding sequence was cloned into pET-21b and pET-32a respectively, to be expressed alone or in the form of fusion protein with thioredoxin (Trx) and His(6)-tag in Escherichia coli BL21 (DE3). The chimeric peptide failed to be expressed alone, but over-expressed in the fusion form, which presented as soluble protein and took up more than 30% of total proteins of host cells. The fusion protein was seriously degraded during the cell disruption, in which endogenous metalloproteinase played a key role. Degradation of target peptide was inhibited by combined application of EDTA in the cell disruption buffer and a step of Source 30Q anion exchange chromatography (AEC) before metal-chelating chromatography (MCAC) for purifying His(6)-tagged fusion protein. The chimeric peptide was recovered from the purified fusion protein by enterokinase digestion at a yield of 3.0 mg/L bacteria culture with a purity of more than 95%. Immunogenicity analysis showed that the recombinant chimeric peptide was able to arouse more than 1×10(4) titers of specific antibody in BALB/c mice. Present work laid a solid foundation for the development of therapeutic peptide vaccine targeting EGFR dimerization and provided a convenient and low-cost preparation method for small peptides.

  15. Different Culture Media Affect Proliferation, Surface Epitope Expression, and Differentiation of Ovine MSC.

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    Adamzyk, Carina; Emonds, Tanja; Falkenstein, Julia; Tolba, René; Jahnen-Dechent, Wilhelm; Lethaus, Bernd; Neuss, Sabine

    2013-01-01

    Orthopedic implants including engineered bone tissue are commonly tested in sheep. To avoid rejection of heterologous or xenogeneic cells, autologous cells are preferably used, that is, ovine mesenchymal stem cells (oMSC). Unlike human MSC, ovine MSC are not well studied regarding isolation, expansion, and characterization. Here we investigated the impact of culture media composition on growth characteristics, differentiation, and surface antigen expression of oMSC. The culture media varied in fetal calf serum (FCS) content and in the addition of supplements and/or additional epidermal growth factor (EGF). We found that FCS strongly influenced oMSC proliferation and that specific combinations of supplemental factors (MCDB-201, ITS-plus, dexamethasone, and L-ascorbic acid) determined the expression of surface epitopes. We compared two published protocols for oMSC differentiation towards the osteogenic, adipogenic, and chondrogenic fate and found (i) considerable donor to donor variations, (ii) protocol-dependent variations, and (iii) variations resulting from the preculture medium composition. Our results indicate that the isolation and culture of oMSC in different growth media are highly variable regarding oMSC phenotype and behaviour. Furthermore, variations from donor to donor critically influence growth rate, surface marker expression, and differentiation.

  16. Different Culture Media Affect Proliferation, Surface Epitope Expression, and Differentiation of Ovine MSC

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    Carina Adamzyk

    2013-01-01

    Full Text Available Orthopedic implants including engineered bone tissue are commonly tested in sheep. To avoid rejection of heterologous or xenogeneic cells, autologous cells are preferably used, that is, ovine mesenchymal stem cells (oMSC. Unlike human MSC, ovine MSC are not well studied regarding isolation, expansion, and characterization. Here we investigated the impact of culture media composition on growth characteristics, differentiation, and surface antigen expression of oMSC. The culture media varied in fetal calf serum (FCS content and in the addition of supplements and/or additional epidermal growth factor (EGF. We found that FCS strongly influenced oMSC proliferation and that specific combinations of supplemental factors (MCDB-201, ITS-plus, dexamethasone, and L-ascorbic acid determined the expression of surface epitopes. We compared two published protocols for oMSC differentiation towards the osteogenic, adipogenic, and chondrogenic fate and found (i considerable donor to donor variations, (ii protocol-dependent variations, and (iii variations resulting from the preculture medium composition. Our results indicate that the isolation and culture of oMSC in different growth media are highly variable regarding oMSC phenotype and behaviour. Furthermore, variations from donor to donor critically influence growth rate, surface marker expression, and differentiation.

  17. RB4CD12 epitope expression and heparan sulfate disaccharide composition in brain vasculature

    NARCIS (Netherlands)

    Hosono-Fukao, T.; Ohtake-Niimi, S.; Nishitsuji, K.; Hossain, M.M.; Kuppevelt, A.H.M.S.M. van; Michikawa, M.; Uchimura, K.

    2011-01-01

    RB4CD12 is a phage display antibody that recognizes a heparan sulfate (HS) glycosaminoglycan epitope. The epitope structure is proposed to contain a trisulfated disaccharide, [-IdoA(2-OSO(3))-GlcNSO(3) (6-OSO(3))-], which supports HS binding to various macromolecules such as growth factors and cytok

  18. Induction of protective immunity in swine by recombinant bamboo mosaic virus expressing foot-and-mouth disease virus epitopes

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    Lin Na-Sheng

    2007-09-01

    Full Text Available Abstract Background Plant viruses can be employed as versatile vectors for the production of vaccines by expressing immunogenic epitopes on the surface of chimeric viral particles. Although several viruses, including tobacco mosaic virus, potato virus X and cowpea mosaic virus, have been developed as vectors, we aimed to develop a new viral vaccine delivery system, a bamboo mosaic virus (BaMV, that would carry larger transgene loads, and generate better immunity in the target animals with fewer adverse environmental effects. Methods We engineered the BaMV as a vaccine vector expressing the antigenic epitope(s of the capsid protein VP1 of foot-and-mouth disease virus (FMDV. The recombinant BaMV plasmid (pBVP1 was constructed by replacing DNA encoding the 35 N-terminal amino acid residues of the BaMV coat protein with that encoding 37 amino acid residues (T128-N164 of FMDV VP1. Results The pBVP1 was able to infect host plants and to generate a chimeric virion BVP1 expressing VP1 epitopes in its coat protein. Inoculation of swine with BVP1 virions resulted in the production of anti-FMDV neutralizing antibodies. Real-time PCR analysis of peripheral blood mononuclear cells from the BVP1-immunized swine revealed that they produced VP1-specific IFN-γ. Furthermore, all BVP1-immunized swine were protected against FMDV challenge. Conclusion Chimeric BaMV virions that express partial sequence of FMDV VP1 can effectively induce not only humoral and cell-mediated immune responses but also full protection against FMDV in target animals. This BaMV-based vector technology may be applied to other vaccines that require correct expression of antigens on chimeric viral particles.

  19. ENDOGENOUS EXPRESSION AND HLA STABILIZATION ASSAY OF PLASMODIUM FALCIPARUM CTL EPITOPE MINIGENE IN HUMAN HLA-A2.1 AND HLA-B51 CELLS

    Institute of Scientific and Technical Information of China (English)

    唐玉阳; 王恒

    2001-01-01

    Objective. To evaluate the Plasmodium falciparum CTL epitope vaccines in HLA class I allele specific human cell lines that have high frequency among Chinese population. Methods. Synthesized oligonucleotides encoding for P.f. CTL epitope genes, constructed eukaryotic expression plasmids, transfected the minigenes into HLA class I allele specific human cell lines and identified endogenous expressing of the minigenes by RT-PCR and HLA stabilization assay. Results. Two mini-genes encoding Plasmodium falciparum CTL epitopes were designed and cloned, respectively, into an eukaryotic expressing vector to form TR26 which was restricted to HLA-B51, SH6 which was restricted to HLA-A2.1, and TS, which had the two aforementioned mini-genes fused in tandem. All of these CTL epitope genes were transfected and endogenously expressed in respective cell lines containing appropriate HLA molecules. The obviously increased expressions of HLA class I molecules were detected in the transfected cell lines. It was demonstrated that the two discrete Plasmodium falciparum epitope genes were effectively processed and presented, and the close proximity of the two epitope genes in one chain as in mini-gene TS did not interfere with the processing and presenting of each epitope gene in corresponding cell line. Conclusion. A successful expression and presentation of multiple CTL epitope mini-gene in MHC class I allele specific human cell lines were demonstrated by an in vitro assay, which could be corresponding to the vaccination of CTL vaccines in people with different MHC I molecules. This work also suggested the possibility of constructing a multiple CTL epitope plasmodium falciparum DNA vaccine that could cover most of Chinese population.

  20. Impact of cell type and epitope tagging on heterologous expression of G protein-coupled receptor: a systematic study on angiotensin type II receptor.

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    Lili Jiang

    Full Text Available Despite heterologous expression of epitope-tagged GPCR is widely adopted for functional characterization, there is lacking of systematic analysis of the impact of expression host and epitope tag on GPCR expression. Angiotensin type II (AT2 receptor displays agonist-dependent and -independent activities, coupling to a spectrum of signaling molecules. However, consensus has not been reached on the subcellular distributions, signaling cascades and receptor-mediated actions. To examine the contributions of host cell and epitope tag on receptor expression and activity, epitope-tagged AT2 receptor variants were transiently or stably expressed in HEK293, CHO-K1 and PC12 cells. The epitope-tagged AT2 receptor variants were detected both on the cell membrane and in the perinuclear region. In transiently transfected HEK293 cells, Myc-AT2 existed predominantly as monomer. Additionally, a ladder of ubiquitinated AT2 receptor proteins was detected. By contrast, stably expressed epitope-tagged AT2 receptor variants existed as both monomer and high molecular weight complexes, and the latter was enriched in cell surface. Glycosylation promoted cell surface expression of Myc-AT2 but had no effect on AT2-GFP in HEK293 cells. In cells that stably expressed Myc-AT2, serum starvation induced apoptosis in CHO-K1 cells but not in HEK293 or PC12 cells. Instead, HEK293 and PC12 cells stably expressing Myc-AT2 exhibited partial cell cycle arrest with cells accumulating at G1 and S phases, respectively. Taken together, these results suggest that expression levels, subcellular distributions and ligand-independent constitutive activities of AT2 receptor were cell type-dependent while posttranslational processing of nascent AT2 receptor protein was modulated by epitope tag and mode of expression.

  1. In silico epitope prediction, expression and functional analysis of Per a 10 allergen from the American cockroach

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    Tong, Xunliang; Guo, Miao; Jin, Min; Chen, Hao; Li, Yanming; Wei, Ji-Fu

    2016-01-01

    Cockroach (CR) allergies caused by the American cockroach hyave been recognized to be repsonsible for IgE-mediated type I hypersensitivity worldwide. Per a 10 is one of the recognized main allergens of the American CR. In a previous study, we examined another American CR allergen, Per a 9 in patients with CR allergies and examined epitope sequences in this allergen. In the present study, we aimed to examine epitope sequences in the Per a 10 allergen. for this purpose, the Per a 10 gene was cloned and expressed in Escherichia coli (E. coli) systems. Our results revealed that 9 out of 16 (56.3%) sera from patients with American CR allergies reacted to Per a10, as assessed by ELISA, confirming that Per a 10 is a major allergen of the American CR. Our results also revealed that the expression of CD63 and CCR3 on passively sensitized basophils (obtained sera of patients with American CR allergies) was increased by approximately 2.3-fold, indicating that recombinant Per a 10 is functionally active. In addition, 3 immunoinformatics tools, namely the DNAStar Protean system, the Bioinformatics Predicted Antigenic Peptides (BPAP) system and the BepiPred 1.0 server were used to predict the peptides and the results revealed 8 peptides (2–12, 55–67, 98–120, 125–133, 149–160, 170–182, 201–208 and 223–227) as potential B cell epitopes of the Per a 10 allergen. Moreover, Per a 10 was predicted to have 3 T cell epitope sequences, namely 83–92, 139–147 and 162–170. The findings of our study on the CR allergen may prove to be useful in the development of peptide-based vaccine for the prevention and/or treatment of CR allergies. PMID:27840898

  2. Expression of Tetanus Toxin Subfragments In Vitro and Characterization of Epitopes

    NARCIS (Netherlands)

    Andersen-Beckh, Bettina; Binz, Thomas; Kurazono, Hisao; Mayer, Thomas; Eisel, Ulrich; Niemann, Heiner

    1989-01-01

    To define epitopes of tetanus toxin, we compared four different in vitro systems in terms of their ability to produce tetanus toxin-specific subfragments from cloned DNA. A transcription-translation system developed from a nontoxigenic strain of Clostridium tetani was found to yield predominantly fu

  3. Identification of Novel HLA-A*24:02-Restricted Epitope Derived from a Homeobox Protein Expressed in Hematological Malignancies.

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    Maiko Matsushita

    Full Text Available The homeobox protein, PEPP2 (RHOXF2, has been suggested as a cancer/testis (CT antigen based on its expression pattern. However, the peptide epitope of PEPP2 that is recognized by cytotoxic T cells (CTLs is unknown. In this study, we revealed that PEPP2 gene was highly expressed in myeloid leukemia cells and some other hematological malignancies. This gene was also expressed in leukemic stem-like cells. We next identified the first reported epitope peptide (PEPP2(271-279. The CTLs induced by PEPP2(271-279 recognized PEPP2-positive target cells in an HLA-A*24:02-restricted manner. We also found that a demethylating agent, 5-aza-2'-deoxycytidine, could enhance PEPP2 expression in leukemia cells but not in blood mononuclear cells from healthy donors. The cytotoxic activity of anti-PEPP2 CTL against leukemic cells treated with 5-aza-2'-deoxycytidine was higher than that directed against untreated cells. These results suggest a clinical rationale that combined treatment with this novel antigen-specific immunotherapy together with demethylating agents might be effective in therapy-resistant myeloid leukemia patients.

  4. High-Level Systemic Expression of Conserved Influenza Epitope in Plants on the Surface of Rod-Shaped Chimeric Particles

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    Natalia V. Petukhova

    2014-04-01

    Full Text Available Recombinant viruses based on the cDNA copy of the tobacco mosaic virus (TMV genome carrying different versions of the conserved M2e epitope from influenza virus A cloned into the coat protein (CP gene were obtained and partially characterized by our group previously; cysteines in the human consensus M2e sequence were changed to serine residues. This work intends to show some biological properties of these viruses following plant infections. Agroinfiltration experiments on Nicotiana benthamiana confirmed the efficient systemic expression of M2e peptides, and two point amino acid substitutions in recombinant CPs significantly influenced the symptoms and development of viral infections. Joint expression of RNA interference suppressor protein p19 from tomato bushy stunt virus (TBSV did not affect the accumulation of CP-M2e-ser recombinant protein in non-inoculated leaves. RT-PCR analysis of RNA isolated from either infected leaves or purified TMV-M2e particles proved the genetic stability of TMV‑based viral vectors. Immunoelectron microscopy of crude plant extracts demonstrated that foreign epitopes are located on the surface of chimeric virions. The rod‑shaped geometry of plant-produced M2e epitopes is different from the icosahedral or helical filamentous arrangement of M2e antigens on the carrier virus-like particles (VLP described earlier. Thereby, we created a simple and efficient system that employs agrobacteria and plant viral vectors in order to produce a candidate broad-spectrum flu vaccine.

  5. Expression and purification of a soluble B lymphocyte stimulator mutant modified with the T-helper cell epitope.

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    Gao, Huiguang; Fu, Weiling; Li, Rongfen; Chen, Linfeng; Ji, Qing; Zhang, Li; Huang, Gang; He, Fengtian

    2006-10-01

    The DNA encoding soluble B lymphocyte stimulator (134-285 amino acids, sBLyS) mutant with residues 217-224 replaced by two glycines (named msBLyS) was constructed. The sequence encoding a foreign immunodominant T-helper epitope from ovalbumin (OVA) was then coupled to the 5'-end of msBLyS cDNA. After being sequenced, the recombinant DNA was ligated into the prokaryotic expression vector pQE-80L. The recombinant protein was produced in E. coli DH5alpha after induction with IPTG with the yield of more than 40% of total bacterial protein. The recombinant protein was purified with Ni-NTA chromatography and Sepharcryl S200 chromatography to a purity of more than 98%. The BALB/c mice, immunized with the recombinant protein, produced anti-BLyS antibodies at a high level, which indicated that the recombinant BLyS mutant modified with T-helper epitope elicited polyclonal antibodies with cross-reactivity with BLyS in vivo. This recombinant protein may therefore be used as immune inhibitor of BLyS for treating BLyS -associated autoimmune diseases.

  6. Transgenic tomatoes express an antigenic polypeptide containing epitopes of the diphtheria, pertussis and tetanus exotoxins, encoded by a synthetic gene.

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    Soria-Guerra, Ruth Elena; Rosales-Mendoza, Sergio; Márquez-Mercado, Crisóforo; López-Revilla, Rubén; Castillo-Collazo, Rosalba; Alpuche-Solís, Angel Gabriel

    2007-07-01

    A current priority of vaccinology is the development of multicomponent vaccines that protect against several pathogens. The diphtheria-pertussis-tetanus (DPT) vaccine prevents the symptoms of three serious and often fatal diseases due to the exotoxins produced by Corynebacterium diphteriae, Bordetella pertussis and Clostridium tetani. We are attempting to develop an edible DPT multicomponent vaccine in plants, based on the fusion of protective exotoxin epitopes encoded by synthetic genes. By means of Agrobacterium mediated transformation we generated transgenic tomatoes with a plant-optimised synthetic gene encoding a novel polypeptide containing two adjuvant and six DPT immunoprotective exotoxin epitopes joined by peptide linkers. In transformed tomato plants, integration of the synthetic DPT (sDPT) gene detected by PCR was confirmed by Southern blot, and specific transcripts of the expected molecular size were detected by RT-PCR. Expression of the putative polypeptide encoded by the sDPT gene was detected by immunoassay with specific antibodies to the diphtheria, pertussis and tetanus exotoxins. The sDPT gene is therefore integrated, transcribed and translated as the expected recombinant sDPT multiepitope polypeptide in transgenic tomatoes that constitute a potential edible vaccine.

  7. Alterations in expression of Cat-315 epitope of perineuronal nets during normal ageing, and its modulation by an open-channel NMDA receptor blocker, memantine.

    Science.gov (United States)

    Yamada, Jun; Ohgomori, Tomohiro; Jinno, Shozo

    2017-03-08

    The perineuronal net (PNN), a specialized aggregate of the extracellular matrix, is involved in neuroprotection against oxidative stress, which is now recognized as a major contributor to age-related decline in brain functions. In this study, we investigated the age-related molecular changes of PNNs using monoclonal antibody Cat-315, which recognizes human natural killer-1 (HNK-1) glycan on aggrecan-based PNNs. Western blot analysis showed that the expression levels of Cat-315 epitope in the hippocampus were higher in middle-aged (MA, 12-month-old) mice than in young adult (YA, 2-month-old) mice. Although there were no differences in the expression levels of Cat-315 epitope between old age (OA, 20-month-old) and MA mice, Cat-315 immunoreactivity was also detected in astrocytes of OA mice. To focus on Cat-315 epitope in PNNs, we used YA and MA mice in the following experiments. Optical disector analysis showed that there were no differences in the numbers of Cat-315-positive (Cat-315(+) ) PNNs between YA and MA mice. Fluorescence intensity analysis indicated that Cat-315 immunoreactivity in PNNs increased with age in the dorsal hippocampus, which is mainly involved in cognitive functions. Administration of an open-channel blocker of NMDA receptor, memantine, reduced the expression levels of Cat-315 epitope in the hippocampus. Furthermore, the numbers of glutamatergic and GABAergic terminals colocalized with Cat-315 epitope around parvalbumin-positive neurons were decreased by memantine. These findings provide novel insight into the involvement of PNNs in normal brain ageing, and suggest that memantine may counteract the age-related alterations in expression levels of Cat-315 epitope via regulation of its subcellular localization. This article is protected by copyright. All rights reserved.

  8. Programmed death-1 expression on HIV-1-specific CD8+ T cells is shaped by epitope specificity, T-cell receptor clonotype usage and antigen load

    DEFF Research Database (Denmark)

    Kløverpris, Henrik N; McGregor, Reuben; McLaren, James E

    2014-01-01

    ) clonotypes within individual HIV-1-specific CD8+ T-cell populations was also apparent, independent of clonal dominance hierarchies. Positive correlations were detected between PD-1 expression and plasma viral load, which were reinforced by stratification for epitope sequence stability and dictated...

  9. B-cell epitope of beta toxin of Clostridium perfringens genetically conjugated to a carrier protein: expression, purification and characterization of the chimeric protein.

    Science.gov (United States)

    Bhatia, Bharti; Solanki, Amit Kumar; Kaushik, Himani; Dixit, Aparna; Garg, Lalit C

    2014-10-01

    Beta toxin (btx) is the prime virulence factor for the pathogenesis of Clostridium perfringens type C strain, known to cause necrotic enteritis and enterotoxaemia in mammalian species. The existing vaccines targeting btx are formaldehyde inactivated culture filtrates of Clostridium. These filtrates raise antigenic load in the host leading to nonspecific and poor responses. The present study aimed to overcome these drawbacks and generate a chimeric protein carrying in silico identified B-cell epitope of btx fused with a carrier protein as a vaccine candidate. Using bioinformatic tools, three stretches of amino acids were predicted as putative B-cell epitopes. One of the epitopes spanning 140-156 amino acid residues was genetically conjugated with B-subunit of heat labile enterotoxin (LTB) of Escherichia coli and expressed as a translational fusion in Vibrio cholerae secretory expression system. High level expression of the recombinant fusion protein rLTB-Btx140-156 was obtained and the protein was successfully purified. The recombinant protein retained the native LTB property to pentamerize and bind to GM1 ganglioside receptor of LTB. The antigenicity of both the epitope and the carrier protein was maintained in fusion protein as indicated by immunoblotting against anti-LTB and anti-btx antibody. The rLTB-Btx140-156 fusion protein therefore can be evaluated as a potential vaccine candidate against C. perfringens.

  10. Adenovirus-mediated expression of pig α(1, 3) galactosyltransferase reconstructs Gal α(1, 3) Gal epitope on the surface of human tumor cells

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    Gal α(1,3)Gal(gal epitope)is a carbohydrate epitope and synthesized in large amount by α(1,3)galactosyltransferase [α(1,3)GT] enzyme on the cells of lower mammalian animals such as pigs and mice.Human has no gal epitope due to the inactivation of α(1,3)GT gene but produces a large amount of antibodies(anti-Gal)which recognize Gal α(1,3)Gal structures specifically.In this study,a replicationdeficient recombinant adenoviral vector Ad5sGT containing pig α(1,3)GT cDNA was constructed and characterized.Adenoviral vector-mediated transfer of pig α(1,3)GT gene into human tumor cells such as malignant melanoma A375,stomach cancer SGC-7901,and lung cancer SPC-A-1 was reported for the first time.Results showed that Gal epitope did not increase the sensitivity of human tumor cells to human complement-mediated lysis,although human complement activation and the binding of human IgG and IgM natural antibodies to human tumor cells were enhanced significantly after Ad5sGT transduction.Appearance of gal epitope on the human tumor cells changed the expression of cell surface carbohydrates reacting with Ulex europaeus I(UEA I)lectins,Vicia villosa agglutinin(VVA),Arachis hypogaea agglutinin(PNA),and Glycine max agglutinin(SBA)to different degrees.In addition,no effect of gal epitope on the growth in vitro of human tumor cells was observed in MTT assay.

  11. Expression and Antigenic Characterization of the Epitope-G1 of the Bovine Ephemeral Fever Virus Glycoprotein in Pichia pastoris

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    The epitope-G1 gene of Bovine ephemeral fever virus (BEFV) glycoprotein was synthesised by PCR and cloned into expression vector pPIC9K to construct recombinant plasmid pPIC9K-G1. Then the pPIC9K-G1 was linearized and transformed into Pichia pastoris GS 115. The recombinant P. pastoris strains were selected by a G418 transformation screen and confirmed by PCR. After being induced with methanol, an expressed protein with 26 kDa molecular weight was obtained, which was much bigger than the predicted size (15.54 kDa). Deglycosylation analysis indicated the recombinant G1 was glycosylated. Western blot and ELISA tests, as well as rabbit immunization and specificity experiments indicated that the target protein had both higher reaction activity and higher immunocompetence and specificity. The recombinant G1 protein could be used as a coating antigen to develop an ELISA kit for bovine ephemeral fever diagnosis.

  12. Dengue virus-infected human dendritic cells reveal hierarchies of naturally expressed novel NS3 CD8 T cell epitopes.

    Science.gov (United States)

    Piazza, P; Campbell, D; Marques, E; Hildebrand, W H; Buchli, R; Mailliard, R; Rinaldo, C R

    2014-09-01

    Detailed knowledge of dengue virus (DENV) cell-mediated immunity is limited. In this study we characterize CD8(+) T lymphocytes recognizing three novel and two known non-structural protein 3 peptide epitopes in DENV-infected dendritic cells. Three epitopes displayed high conservation (75-100%), compared to the others (0-50%). A hierarchy ranking based on magnitude and polyfunctionality of the antigen-specific response showed that dominant epitopes were both highly conserved and cross-reactive against multiple DENV serotypes. These results are relevant to DENV pathogenesis and vaccine design.

  13. A HLA-A2 restricted human CTL line recognizes a novel tumor cell expressed p53 epitope

    DEFF Research Database (Denmark)

    Würtzen, Peter A; Claesson, Mogens H

    2002-01-01

    , the CTL line, which expressed relatively low affinity for the HLA-A2/peptide complex, was able to kill 3 different HLA-A2(+) p53 mutated tumor cell lines. The present and our previous observations expand the number of p53-derived peptides suitable for vaccination protocols for cancer patients with p53......A p53 peptide-specific CTL line was generated through stimulation with autologous monocyte-derived dendritic cells (DC) pulsed with wild-type HLA-A2 binding p53 derived peptides. A p53 peptide-specific CD8(+) CTL line was established from a healthy HLA-A2 positive donor. The CTL line...... was characterized with respect to specificity, affinity and killing of cell lines derived from p53 mutated spontaneous tumors. The CTL line demonstrated lysis of p53(139-147) pulsed target cells and cold target inhibition experiments as well as antibody blocking confirmed that the killing was epitope-specific, HLA...

  14. Induction of mucosal immunity by intranasal immunization with recombinant adenovirus expressing major epitopes of Porcine circovirus-2 capsid protein.

    Science.gov (United States)

    Liu, Yu-feng; Guo, Quan-hai; Chen, Lu; Zhao, Jun; Chang, Hong-tao; Wang, Xin-wei; Yang, Xia; Wang, Chuan-qing

    2013-07-15

    Porcine circovirus-2 (PCV-2) is primarily transmitted through mucosa, thus the mucosal immunity may constitute an essential feature of vaccination strategies against PCV-2 infection. Mucosal immunity elicited by recombinant replication-deficient adenovirus expressing the major epitopes of PCV-2 capsid protein (rAd/Cap/518) via intranasal (i.n.), intramuscular (i.m.) or oral routes in mice were evaluated. Immunization with rAd/Cap/518 via i.n. route induced higher titers of IgA in saliva, bronchoalveolar and intestinal lavage fluid compared with those immunized via i.m. route. The proportions of CD3+, CD3+CD4+ and CD3+CD8+ T cells were significantly increased in mice immunized with rAd/Cap/518 via i.n. route compared with the control group. Higher levels of IFN-γ were detected in the spleen and mesenteric lymph nodes of mice immunized with rAd/Cap/518 via i.n. route compared with other groups, yet IL-4 was not detected in any group. Real-time PCR analysis confirmed viral DNA loads in the i.m. or i.n. immunization group was lower than that seen in the rAd immunization. These results indicate that i.n. administration of rAd/Cap/518 can elicit humoral and Th1-type cellular protective immunity in both systemic and mucosal immune compartments in mice, representing a promising mucosal vaccine candidate against PCV-2.

  15. Construction of prokaryotic expression system of TGF-β1 epitope gene and identification of recombinant fusion protein immunity

    Institute of Scientific and Technical Information of China (English)

    Yong-Hong Guo; Zhi-Ming Hao; Jin-Yan Luo; Jun-Hong Wang

    2005-01-01

    AIM: To insert the constructed TGF-β1the el loop of C-terminus of truncated hepatitis B core antigen to increase TGF-β1expression system and to identify immunity of the expressed recombinant protein in order to exploit the possibility for obtaining anti- TGF-β1METHODS: The TGF-β1mature TGF-β1TGF-32) was amplified by polymerase chain reaction from the recombinant pGEM-7z/TGF-β1HBcAg gene fragments (encoding HBcAg from 1-71 and 89-144 amino acid residues) were amplified from PYTA1-HBcAg vector. The recombinant vector pGEMEX-1 was used to insert HBcAg1-71, TGF-β1into restrictive endonuclease enzyme and ligated with T4ligase. The fusion gene fragments HBcAg1-71-TGF-β1HBcAg89-144 were recloned to pET28a(+) and the DNA sequence was confirmed by the dideoxy chain termination method. The recombinant vector pET28a (+)/CTC was transformed and expressed in E.. Coli BL21 (DE3)under induction of IPTG. After purification with Ni+2-NTA agarose resins, the antigenicity of purified protein was detected by ELISA and Western blot and visualized under electron microscope.RESULTS: Enzyme digestion analysis and sequencing showed that TGF-β1loop of C-terminus of truncated hepatitis B core antigen.SDS-PAGE analysis showed that relative molecular mass(Mr) of the expressed product by pET28a (+)/CTC was Mr 24 600.The output of the target recombinant protein was approximately 34.8% of the total bacterial protein,mainly presented in the form of inclusion body. Western blotting and ELISA demonstrated that the fusion protein could combine with anti-TGF-β1not with anti-HBcAg. The purity of protein was about 90% and the protein was in the form of self-assembling particles visualized under electron microscope. This fusion protein had good anti-TGF-β1could be used as anti-TGF-β1CONCLUSION: A recombinant prokaryotic expression system with high expression efficiency of the target TGF- epitope gene was successfully established.The fusion protein is in the form of self-assembling particles

  16. A Sulfated Glycosaminoglycan Linkage Region is a Novel Type of Human Natural Killer-1 (HNK-1 Epitope Expressed on Aggrecan in Perineuronal Nets.

    Directory of Open Access Journals (Sweden)

    Keiko Yabuno

    Full Text Available Human natural killer-1 (HNK-1 carbohydrate (HSO3-3GlcAβ1-3Galβ1-4GlcNAc-R is highly expressed in the brain and required for learning and neural plasticity. We previously demonstrated that expression of the HNK-1 epitope is mostly abolished in knockout mice for GlcAT-P (B3gat1, a major glucuronyltransferase required for HNK-1 biosynthesis, but remained in specific regions such as perineuronal nets (PNNs in these mutant mice. Considering PNNs are mainly composed of chondroitin sulfate proteoglycans (CSPGs and regulate neural plasticity, GlcAT-P-independent expression of HNK-1 in PNNs is suggested to play a role in neural plasticity. However, the function, structure, carrier glycoprotein and biosynthetic pathway for GlcAT-P-irrelevant HNK-1 epitope remain unclear. In this study, we identified a unique HNK-1 structure on aggrecan in PNNs. To determine the biosynthetic pathway for the novel HNK-1, we generated knockout mice for GlcAT-S (B3gat2, the other glucuronyltransferase required for HNK-1 biosynthesis. However, GlcAT-P and GlcAT-S double-knockout mice did not exhibit reduced HNK-1 expression compared with single GlcAT-P-knockout mice, indicating an unusual biosynthetic pathway for the HNK-1 epitope in PNNs. Aggrecan was purified from cultured cells in which GlcAT-P and -S are not expressed and we determined the structure of the novel HNK-1 epitope using liquid chromatography/mass spectrometry (LC/MS as a sulfated linkage region of glycosaminoglycans (GAGs, HSO3-GlcA-Gal-Gal-Xyl-R. Taken together, we propose a hypothetical model where GlcAT-I, the sole glucuronyltransferase required for synthesis of the GAG linkage, is also responsible for biosynthesis of the novel HNK-1 on aggrecan. These results could lead to discovery of new roles of the HNK-1 epitope in neural plasticity.

  17. Molecular cloning, expression, IgE binding activities and in silico epitope prediction of Per a 9 allergens of the American cockroach

    Science.gov (United States)

    Yang, Haiwei; Chen, Hao; Jin, Min; Xie, Hua; He, Shaoheng; Wei, Ji-Fu

    2016-01-01

    Per a 9 is a major allergen of the American cockroach (CR), which has been recognized as an important cause of imunoglobulin E-mediated type I hypersensitivity worldwide. However, it is not neasy to obtain a substantial quantity of this allergen for use in functional studies. In the present study, the Per a 9 gene was cloned and expressed in Escherichia coli (E. coli) systems. It was found that 13/16 (81.3%) of the sera from patients with allergies caused by the American CR reacted to Per a 9, as assessed by enzyme-linked immunosorbent assay, confirming that Per a 9 is a major allergen of CR. The induction of the expression of CD63 and CCR3 in passively sensitized basophils (from sera of patients with allergies caused by the American CR) by approximately 4.2-fold indicated that recombinant Per a 9 was functionally active. Three immunoinformatics tools, including the DNASTAR Protean system, Bioinformatics Predicted Antigenic Peptides (BPAP) system and the BepiPred 1.0 server were used to predict the potential B cell epitopes, while Net-MHCIIpan-2.0 and NetMHCII-2.2 were used to predict the T cell epitopes of Per a 9. As a result, we predicted 11 peptides (23–28, 39–46, 58–64, 91–118, 131–136, 145–154, 159–165, 176–183, 290–299, 309–320 and 338–344) as potential B cell linear epitopes. In T cell prediction, the Per a 9 allergen was predicted to have 5 potential T cell epitope sequences, 119–127, 194–202, 210–218, 239–250 and 279–290. The findings of our study may prove to be useful in the development of peptide-based vaccines to combat CR-induced allergies. PMID:27840974

  18. Transient expression of Human papillomavirus type 16 L2 epitope fused to N- and C-terminus of coat protein of Potato virus X in plants

    Indian Academy of Sciences (India)

    Noemi Cerovska; Hana Hoffmeisterova; Tomas Moravec; Helena Plchova; Jitka Folwarczna; Helena Synkova; Helena Ryslava; Viera Ludvikova; Michal Smahel

    2012-03-01

    Transient expression of foreign genes based on plant viral vectors is a suitable system for the production of relevant immunogens that can be used for the development of a new generation of vaccines against a variety of infectious diseases. In the present study the epitope derived from HPV-16 L2 minor capsid protein (amino acids 108–120) was expressed from Potato virus X (PVX)-based vector pGR106 as N- or C-terminal fusion with the PVX coat protein (PVX CP) in transgenic Nicotiana benthamiana plants. The fusion protein L2108-120-PVX CP was successfully expressed in plants at a level of 170 mg/kg of fresh leaf tissue. The C-terminal fusion protein PVX CP- L2108-120 was expressed using mutated vector sequence to avoid homologous recombination at a level of 8 mg/kg of fresh leaf tissue. Immunogenicity of L2108-120-PVX CP virus-like particles was tested after immunization of mice by subcutaneous injection or tattoo administration. In animal sera the antibodies against the PVX CP and the L2108-120 epitope were found after both methods of vaccine delivery.

  19. Molecular cloning, expression, IgE binding activities and in silico epitope prediction of Per a 9 allergens of the American cockroach.

    Science.gov (United States)

    Yang, Haiwei; Chen, Hao; Jin, Min; Xie, Hua; He, Shaoheng; Wei, Ji-Fu

    2016-12-01

    Per a 9 is a major allergen of the American cockroach (CR), which has been recognized as an important cause of imunoglobulin E-mediated type I hypersensitivity worldwide. However, it is not neasy to obtain a substantial quantity of this allergen for use in functional studies. In the present study, the Per a 9 gene was cloned and expressed in Escherichia coli (E. coli) systems. It was found that 13/16 (81.3%) of the sera from patients with allergies caused by the American CR reacted to Per a 9, as assessed by enzyme-linked immunosorbent assay, confirming that Per a 9 is a major allergen of CR. The induction of the expression of CD63 and CCR3 in passively sensitized basophils (from sera of patients with allergies caused by the American CR) by approximately 4.2-fold indicated that recombinant Per a 9 was functionally active. Three immunoinformatics tools, including the DNAStar Protean system, Bioinformatics Predicted Antigenic Peptides (BPAP) system and the BepiPred 1.0 server were used to predict the potential B cell epitopes, while Net-MHCIIpan-2.0 and NetMHCII-2.2 were used to predict the T cell epitopes of Per a 9. As a result, we predicted 11 peptides (23-28, 39-46, 58-64, 91-118, 131-136, 145-154, 159-165, 176-183, 290-299, 309-320 and 338-344) as potential B cell linear epitopes. In T cell prediction, the Per a 9 allergen was predicted to have 5 potential T cell epitope sequences, 119-127, 194-202, 210-218, 239-250 and 279-290. The findings of our study may prove to be useful in the development of peptide-based vaccines to combat CR-induced allergies.

  20. Modules for C-terminal epitope tagging of Tetrahymena genes

    Science.gov (United States)

    Kataoka, Kensuke; Schoeberl, Ursula E.; Mochizuki, Kazufumi

    2010-01-01

    Although epitope tagging has been widely used for analyzing protein function in many organisms, there are few genetic tools for epitope tagging in Tetrahymena. In this study, we describe several C-terminal epitope tagging modules that can be used to express tagged proteins in Tetrahymena cells by both plasmid- and PCR-based strategies. PMID:20624430

  1. Reversal of tolerance induced by transplantation of skin expressing the immunodominant T cell epitope of rat type II collagen entitles development of collagen-induced arthritis but not graft rejection

    DEFF Research Database (Denmark)

    Bäcklund, Johan; Treschow, Alexandra; Firan, Mihail

    2002-01-01

    collagen (CI), e.g. in skin, are tolerized against rat CII and resistant to CIA. In this study we transplanted skin from TSC transgenic mice onto non-transgenic CIA-susceptible littermates to investigate whether introduction of this epitope to a naïve immune system would lead to T cell priming and graft......Collagen-induced arthritis (CIA) is induced in H-2(q) mice after immunization with rat type II collagen (CII). The immunodominant T cell epitope on heterologous CII has been located to CII256-270. We have previously shown that TSC transgenic mice, which express the heterologous epitope in type I...... rejection or instead to tolerance and arthritis protection. Interestingly, TSC grafts were accepted and not even immunization of recipient mice with CII in adjuvant induced graft rejection. Instead, TSC skin recipients displayed a reduced T and B cell response to CII and were also protected from arthritis...

  2. Expression of a multi-epitope DPT fusion protein in transplastomic tobacco plants retains both antigenicity and immunogenicity of all three components of the functional oligomer.

    Science.gov (United States)

    Soria-Guerra, Ruth Elena; Alpuche-Solís, Angel G; Rosales-Mendoza, Sergio; Moreno-Fierros, Leticia; Bendik, Elise M; Martínez-González, Luzmila; Korban, Schuyler S

    2009-05-01

    Expression of genes in plant chloroplasts provides an opportunity for enhanced production of target proteins. We report the introduction and expression of a fusion DPT protein containing immunoprotective exotoxin epitopes of Corynebacterium diphtheriae, Bordetella pertussis, and Clostridium tetani in tobacco chloroplasts. Using biolistic-mediated transformation, a plant-optimized synthetic DPT gene was successfully transferred to tobacco plastomes. Putative transplastomic T0 plants were identified by PCR, and Southern blot analysis confirmed homoplasmy in T1 progeny. ELISA assays demonstrated that the DPT protein retained antigenicity of the three components of the fusion protein. The highest level of expression in these transplastomic plants reached 0.8% of total soluble protein. To assess whether the functional recombinant protein expressed in tobacco plants would induce specific antibodies in test animals, a mice feeding experiment was conducted. For mice orally immunized with freeze-dried transplastomic leaves, production of IgG and IgA antibodies specific to each toxin were detected in serum and mucosal tissues.

  3. Expression of toxin co-regulated pilus subunit A (TCPA) of Vibrio cholerae and its immunogenic epitopes fused to cholera toxin B subunit in transgenic tomato (Solanum lycopersicum).

    Science.gov (United States)

    Sharma, Manoj Kumar; Singh, Nirmal Kumar; Jani, Dewal; Sisodia, Rama; Thungapathra, M; Gautam, J K; Meena, L S; Singh, Yogendra; Ghosh, Amit; Tyagi, Akhilesh Kumar; Sharma, Arun Kumar

    2008-02-01

    For protection against cholera, it is important to develop efficient vaccine capable of inducing anti-toxin as well as anti-colonizing immunity against Vibrio cholerae infections. Earlier, expression of cholera toxin B subunit (CTB) in tomato was reported by us. In the present investigation, toxin co-regulated pilus subunit A (TCPA), earlier reported to be an antigen capable of providing anti-colonization immunity, has been expressed in tomato. Further, to generate more potent combinatorial antigens, nucleotides encoding P4 or P6 epitope of TCPA were fused to cholera toxin B subunit gene (ctxB) and expressed in tomato. Presence of transgenes in the tomato genome was confirmed by PCR and expression of genes was confirmed at transcript and protein level. TCPA, chimeric CTB-P4 and CTB-P6 proteins were also expressed in E. coli. TCPA protein expressed in E. coli was purified to generate anti-TCPA antibodies in rabbit. Immunoblot and G(M1)-ELISA verified the synthesis and assembly of pentameric chimeric proteins in fruit tissue of transgenic tomato plants. The chimeric protein CTB-P4 and CTB-P6 accumulated up to 0.17 and 0.096% of total soluble protein (TSP), respectively, in tomato fruits. Whereas expression of TCPA, CTB-P4 and CTB-P6 in E. coli can be utilized for development of conventional vaccine, expression of these antigens which can provide both anti-toxin as well as anti-colonization immunity, has been demonstrated in plants, in a form which is potentially capable of inducing immune response against cholera infection.

  4. Cloning of linear T-cell epitops of EG95 antigen of Echinococcus granulosus into pGEX4t1 vector and expression analysis by SDS-PAGE

    Directory of Open Access Journals (Sweden)

    Roghayeh Mesri

    2016-12-01

    Full Text Available Background & Objectives: Echinococcus granulosus causes a common disease between humans and animals that called hydatid cyst or hydatidosis. The recombinant EG95 vaccine based on cloning of the 25 KD oncospheral antigen-Eg95- in pGEX stimulated significant humoral and cellular immunity in sheep, but regarding the effect of TH1cell immunity against this parasite and the influence of the linear T-cell epitopes in stimulating this immunity, only the coding sequence of linear T Cell epitopes of EG95 was cloned in pGEX and analyzed it's expression to optimize the qualification of this available vaccine in this study. Materials & Methods: The coding sequence of EG95 linear epitopes by IEDB software were predicted and synthesized. After PCR, the amplicon and pGEX4T1 were digested by xhoI restriction enzyme, the fragment was cloned into pGEX4T1 by heat shock method, and Positive colonies were selected by direct PCR with specific primers. The recombinant protein expression was evaluated in BL21 cells by 10% SDS-PAGE. Results: The coding sequence of EG95 linear T-cell epitopes was amplified by PCR and cloned into pGEX4T1 vector.  The recombinant plasmid was selected with Colony PCR and size difference between intact and recombinant purificated vectors. Recombinant protein expression with high significant concentration was recognized by SDS-PAGE. Conclusion: The EG95 linear T-cell epitopes coding sequence has been successfully cloned into pGEX4T1 vector and expressed into BL21 cells.

  5. Epitopes of anti-RIFIN antibodies and characterization of rif-expressing Plasmodium falciparum parasites by RNA sequencing

    Science.gov (United States)

    Ch’ng, Jun-Hong; Sirel, Madle; Zandian, Arash; del Pilar Quintana, Maria; Chun Leung Chan, Sherwin; Moll, Kirsten; Tellgren-Roth, Asa; Nilsson, IngMarie; Nilsson, Peter; Qundos, Ulrika; Wahlgren, Mats

    2017-01-01

    Variable surface antigens of Plasmodium falciparum have been a major research focus since they facilitate parasite sequestration and give rise to deadly malaria complications. Coupled with its potential use as a vaccine candidate, the recent suggestion that the repetitive interspersed families of polypeptides (RIFINs) mediate blood group A rosetting and influence blood group distribution has raised the research profile of these adhesins. Nevertheless, detailed investigations into the functions of this highly diverse multigene family remain hampered by the limited number of validated reagents. In this study, we assess the specificities of three promising polyclonal anti-RIFIN antibodies that were IgG-purified from sera of immunized animals. Their epitope regions were mapped using a 175,000-peptide microarray holding overlapping peptides of the P. falciparum variable surface antigens. Through immunoblotting and immunofluorescence imaging, we show that different antibodies give varying results in different applications/assays. Finally, we authenticate the antibody-based detection of RIFINs in two previously uncharacterized non-rosetting parasite lines by identifying the dominant rif transcripts using RNA sequencing. PMID:28233866

  6. Expression of Alzheimer-type Neurofibrillary Epitopes in Primary Rat Cortical Neurons Following Infection with Enterococcus faecalis

    Directory of Open Access Journals (Sweden)

    Robert eUnderly

    2016-01-01

    Full Text Available The neurofibrillary tau pathology and amyloid deposits seen in Alzheimer's disease (AD also have been seen in bacteria-infected brains. However, few studies have examined the role of these bacteria in the generation of tau pathology. One suggested link between infection and Alzheimer’s disease is edentulism, the complete loss of teeth. Edentulism can result from chronic periodontal disease due to infection by Enterococcus faecalis. The current study assessed the ability to generate early Alzheimer-like neurofibrillary epitopes in primary rat cortical neurons through bacterial infection by Enterococcus faecalis. Seven-day old cultured neurons were infected with Enterococcus faecalis for 24- and 48-hours. An upward molecular weight shift in tau by western blotting and increased appearance of tau reactivity in cell bodies and degenerating neurites was found in the 48-hour infection group for the antibody CP13 (phospho-Serine-202. A substantial increase in reactivity of Alz-50 was seen at 24- and 48- hours after infection. Furthermore, extensive MAP2 reactivity also was seen at 24- and 48-hours post-infection. Our preliminary findings suggest a potential link between Enterococcus faecalis infection and intracellular changes that may help facilitate early AD-like neurofibrillary pathology.

  7. Generation of human scFvs antibodies recognizing a prion protein epitope expressed on the surface of human lymphoblastoid cells

    Directory of Open Access Journals (Sweden)

    Imperiale Valentina

    2007-07-01

    Full Text Available Abstract Background A hallmark of prion disease is the transformation of normal cellular prion protein (PrPc into an infectious disease-associated isoform, (PrPsc. Anti-prion protein monoclonal antibodies are invaluable for structure-function studies of PrP molecules. Furthermore recent in vitro and in vivo studies indicate that anti-PrP monoclonal antibodies can prevent the incorporation of PrPc into propagating prions. In the present article, we show two new human phage antibodies, isolated on recombinant hamster prion protein (rHaPrP. Results We adopted an antibody phage display strategy to isolate specific human antibodies directed towards rHaPrP which has been used as a bait for panning the synthetic ETH-2 antibody phage library. Two phage antibodies clones named MA3.B4 and MA3.G3 were isolated and characterized under genetic biochemical and immunocytochemical aspects. The clones were found to recognize the prion protein in ELISA studies. In flow-cytometry studies, these human single chain Fragment variable (scFv phage-antibodies show a well defined pattern of reactivity on human lymphoblastoid and myeloid cells. Conclusion Sequence analysis of the gene encoding for the antibody fragments and antigen recognition patterns determined by flow-cytometry analysis indicate that the isolated scFvs recognize novel epitopes in the PrPc molecule. These new anti PrPc human antibodies are unique reagents for prion protein detection and may represent a biologic platform to develop new reagents to treat PrPsc associated disease.

  8. Laminin alpha2 chain-deficient congenital muscular dystrophy: variable epitope expression in severe and mild cases

    DEFF Research Database (Denmark)

    Cohn, R D; Herrmann, R; Sorokin, L;

    1998-01-01

    To characterize the expression of distinct fragments of laminin alpha2 chain in patients with partial laminin alpha2 chain deficiency and variable clinical severity.......To characterize the expression of distinct fragments of laminin alpha2 chain in patients with partial laminin alpha2 chain deficiency and variable clinical severity....

  9. Functional expression of human NKCC1 from a synthetic cassette-based cDNA: introduction of extracellular epitope tags and removal of cysteines.

    Directory of Open Access Journals (Sweden)

    Suma Somasekharan

    Full Text Available The Na-K-Cl cotransporter (NKCC couples the movement of Na(+, K(+, and Cl(- ions across the plasma membrane of most animal cells and thus plays a central role in cellular homeostasis and human physiology. In order to study the structure, function, and regulation of NKCC1 we have engineered a synthetic cDNA encoding the transporter with 30 unique silent restriction sites throughout the open reading frame, and with N-terminal 3xFlag and YFP tags. We show that the novel cDNA is appropriately expressed in HEK-293 cells and that the YFP-tag does not alter the transport function of the protein. Utilizing the Cl(- -sensing capability of YFP, we demonstrate a sensitive assay of Na-K-Cl cotransport activity that measures normal cotransport activity in a fully activated transporter. In addition we present three newly developed epitope tags for NKCC1 all of which can be detected from outside of the cell, one of which is very efficiently delivered to the plasma membrane. Finally, we have characterized cysteine mutants of NKCC1 and found that whereas many useful combinations of cysteine mutations are tolerated by the biosynthetic machinery, the fully "cys-less" NKCC1 is retained in the endoplasmic reticulum. Together these advances are expected to greatly assist future studies of NKCC1.

  10. Functional expression of human NKCC1 from a synthetic cassette-based cDNA: introduction of extracellular epitope tags and removal of cysteines.

    Science.gov (United States)

    Somasekharan, Suma; Monette, Michelle Y; Forbush, Biff

    2013-01-01

    The Na-K-Cl cotransporter (NKCC) couples the movement of Na(+), K(+), and Cl(-) ions across the plasma membrane of most animal cells and thus plays a central role in cellular homeostasis and human physiology. In order to study the structure, function, and regulation of NKCC1 we have engineered a synthetic cDNA encoding the transporter with 30 unique silent restriction sites throughout the open reading frame, and with N-terminal 3xFlag and YFP tags. We show that the novel cDNA is appropriately expressed in HEK-293 cells and that the YFP-tag does not alter the transport function of the protein. Utilizing the Cl(-) -sensing capability of YFP, we demonstrate a sensitive assay of Na-K-Cl cotransport activity that measures normal cotransport activity in a fully activated transporter. In addition we present three newly developed epitope tags for NKCC1 all of which can be detected from outside of the cell, one of which is very efficiently delivered to the plasma membrane. Finally, we have characterized cysteine mutants of NKCC1 and found that whereas many useful combinations of cysteine mutations are tolerated by the biosynthetic machinery, the fully "cys-less" NKCC1 is retained in the endoplasmic reticulum. Together these advances are expected to greatly assist future studies of NKCC1.

  11. Screening of transgenic proteins expressed in transgenic food crops for the presence of short amino acid sequences identical to potential, IgE – binding linear epitopes of allergens

    Directory of Open Access Journals (Sweden)

    Peijnenburg Ad ACM

    2002-12-01

    Full Text Available Abstract Background Transgenic proteins expressed by genetically modified food crops are evaluated for their potential allergenic properties prior to marketing, among others by identification of short identical amino acid sequences that occur both in the transgenic protein and allergenic proteins. A strategy is proposed, in which the positive outcomes of the sequence comparison with a minimal length of six amino acids are further screened for the presence of potential linear IgE-epitopes. This double track approach involves the use of literature data on IgE-epitopes and an antigenicity prediction algorithm. Results Thirty-three transgenic proteins have been screened for identities of at least six contiguous amino acids shared with allergenic proteins. Twenty-two transgenic proteins showed positive results of six- or seven-contiguous amino acids length. Only a limited number of identical stretches shared by transgenic proteins (papaya ringspot virus coat protein, acetolactate synthase GH50, and glyphosate oxidoreductase and allergenic proteins could be identified as (part of potential linear epitopes. Conclusion Many transgenic proteins have identical stretches of six or seven amino acids in common with allergenic proteins. Most identical stretches are likely to be false positives. As shown in this study, identical stretches can be further screened for relevance by comparison with linear IgE-binding epitopes described in literature. In the absence of literature data on epitopes, antigenicity prediction by computer aids to select potential antibody binding sites that will need verification of IgE binding by sera binding tests. Finally, the positive outcomes of this approach warrant further clinical testing for potential allergenicity.

  12. Screening of transgenic proteins expressed in transgenic food crops for the presence of short amino acid sequences identical to potential, IgE – binding linear epitopes of allergens

    Science.gov (United States)

    Kleter, Gijs A; Peijnenburg, Ad ACM

    2002-01-01

    Background Transgenic proteins expressed by genetically modified food crops are evaluated for their potential allergenic properties prior to marketing, among others by identification of short identical amino acid sequences that occur both in the transgenic protein and allergenic proteins. A strategy is proposed, in which the positive outcomes of the sequence comparison with a minimal length of six amino acids are further screened for the presence of potential linear IgE-epitopes. This double track approach involves the use of literature data on IgE-epitopes and an antigenicity prediction algorithm. Results Thirty-three transgenic proteins have been screened for identities of at least six contiguous amino acids shared with allergenic proteins. Twenty-two transgenic proteins showed positive results of six- or seven-contiguous amino acids length. Only a limited number of identical stretches shared by transgenic proteins (papaya ringspot virus coat protein, acetolactate synthase GH50, and glyphosate oxidoreductase) and allergenic proteins could be identified as (part of) potential linear epitopes. Conclusion Many transgenic proteins have identical stretches of six or seven amino acids in common with allergenic proteins. Most identical stretches are likely to be false positives. As shown in this study, identical stretches can be further screened for relevance by comparison with linear IgE-binding epitopes described in literature. In the absence of literature data on epitopes, antigenicity prediction by computer aids to select potential antibody binding sites that will need verification of IgE binding by sera binding tests. Finally, the positive outcomes of this approach warrant further clinical testing for potential allergenicity. PMID:12477382

  13. An antibody produced in tobacco expressing a hybrid beta-1,4-galactosyltransferase is essentially devoid of plant carbohydrate epitopes

    NARCIS (Netherlands)

    H. Bakker; G.J.A. Rouwendal; A.S. Karnoup; D.E.A. Florack; G.M. Stoopen; J.P.F.G. Helsper; R. van Ree; I. van Die; D. Bosch

    2006-01-01

    N-glycosylation of a mAb may have a major impact on its therapeutic merits. Here, we demonstrate that expression of a hybrid enzyme (called xylGaIT), consisting of the N-terminal domain of Arabidopsis thaliana xylosyltra nsf erase and the catalytic domain of human 0-1,4-galactosyltransf erase I (GaI

  14. Antigen epitope of Helicobacter pylorivacuolating cytotoxin A

    Institute of Scientific and Technical Information of China (English)

    Xiu-Li Liu; Shu-Qin Li; Chun-Jie Liu; Hao-Xia Tao; Zhao-Shan Zhang

    2004-01-01

    AIM: To construct and select antigen epitopes of vacuolating cytotoxin A (VacA) for nontoxic VacA vaccine against Helicobacter pylori (H pylori) infection.METHODS: Eleven VacA epitopes were predicted according to VacA antigenic bioinformatics. Three candidates of VacA epitope were constructed through different combined epitopes. The candidate was linked with E. coli heat-labile enterotoxin B (LTB) by a linker of 7 amino acids, and cloned into plasmid pQE-60 in which fusion LTB-VacA epitope was efficiently expressed. To test the antigencity of the candidate, 6 BALB/c mice were treated with the fusion LTB-VacA epitope through intraperitoneal injection. To explore the ability of inhibiting the toxicity of VacA, cantiserum against the candidate was used to counteract VacA that induced HeLa cells to produce cell vacuoles in vitro.RESULTS: Serum IgG against the candidate was induced in the BALB/c mice. In vitro, the three antisera against the candidate efficiently counteracted the toxicity of VacA, and decreased the number of cell vacuoles by 14.17%, 20.20%and 30.41% respectively.CONCLUSION: Two of the three candidates, LZ-VacA1and LZ-VacA2, can be used to further study the mechanism of vacuolating toxicity of VacA, and to construct nontoxic VacA vaccine against H pylori infection.

  15. A cytomegalovirus-based vaccine expressing a single tumor-specific CD8+ T-cell epitope delays tumor growth in a murine model of prostate cancer.

    Science.gov (United States)

    Klyushnenkova, Elena N; Kouiavskaia, Diana V; Parkins, Christopher J; Caposio, Patrizia; Botto, Sara; Alexander, Richard B; Jarvis, Michael A

    2012-06-01

    Cytomegalovirus (CMV) is a highly immunogenic virus that results in a persistent, life-long infection in the host typically with no ill effects. Certain unique features of CMV, including its capacity to actively replicate in the presence of strong host CMV-specific immunity, may give CMV an advantage compared with other virus-based vaccine delivery platforms. In the present study, we tested the utility of mouse CMV (mCMV)-based vaccines expressing human prostate-specific antigen (PSA) for prostate cancer immunotherapy in double-transgenic mice expressing PSA and HLA-DRB1*1501 (DR2bxPSA F1 mice). We assessed the capacity of 2 mCMV-based vectors to induce PSA-specific CD8 T-cell responses and affect the growth of PSA-expressing Transgenic Adenocarcinoma of the Mouse Prostate tumors (TRAMP-PSA). In the absence of tumor challenge, immunization with mCMV vectors expressing either a H2-D(b)-restricted epitope PSA(65-73) (mCMV/PSA(65-73)) or the full-length gene for PSA (mCMV/PSA(FL)) induced comparable levels of CD8 T-cell responses that increased (inflated) with time. Upon challenge with TRAMP-PSA tumor cells, animals immunized with mCMV/PSA(65-73) had delay of tumor growth and increased PSA-specific CD8 T-cell responses, whereas animals immunized with mCMV/PSA(FL) showed progressive tumor growth and no increase in number of splenic PSA(65-73)-specific T cells. The data show that a prototype CMV-based prostate cancer vaccine can induce an effective antitumor immune response in a "humanized" double-transgenic mouse model. The observation that mCMV/PSA(FL) is not effective against TRAMP-PSA is consistent with our previous findings that HLA-DRB1*1501-restricted immune responses to PSA are associated with suppression of effective CD8 T-cell responses to TRAMP-PSA tumors.

  16. Enhancing mucosal immunity in mice by recombinant adenovirus expressing major epitopes of porcine circovirus-2 capsid protein delivered with cytosine-phosphate-guanosine oligodeoxynucleotides.

    Science.gov (United States)

    Chang, Hong-Tao; He, Xiu-Yuan; Liu, Yu-Feng; Chen, Lu; Guo, Quan-Hai; Yu, Qiu-Ying; Zhao, Jun; Wang, Xin-Wei; Yang, Xia; Wang, Chuan-Qing

    2014-01-01

    A recombinant replication-defective adenovirus expressing the major epitopes of porcine circovirus-2 (PCV-2) capsid protein (rAd/Cap/518) was previously constructed and shown to induce mucosal immunity in mice following intranasal delivery. In the present study, immune responses induced by intranasal immunization with a combination of rAd/Cap/518 and cytosine-phosphate-guanosine oligodeoxynucleotides (CpG ODN) were evaluated in mice. The levels of PCV-2-specific IgG in serum and IgA in saliva, lung, and intestinal fluids were significantly higher in the group immunized with rAd/Cap/518 and CpG ODN than animals immunized with rAd/Cap/518 alone. The frequencies of IL-2-secreting CD4⁺ T cells and IFN-γ-producing CD8⁺ T cells were significantly higher in the combined immunization group than mice immunized with rAd/Cap/518 alone. The frequencies of CD3⁺, CD3⁺CD4⁺CD8⁻, and CD3⁺CD4⁻CD8⁺ T cells in the combined immunization group were similar to that treated with CpG ODN alone, but significantly higher than mice that did not receive CpG ODN. PCV-2 load after challenge in the combined immunization group was significantly lower than that in the phosphate-buffered saline placebo group and approximately 7-fold lower in the group treated with CpG ODN alone. These results indicate that rAd/Cap/518 combined with CpG ODN can enhance systemic and local mucosal immunity in mice, and represent a promising synergetic mucosal vaccine against PCV-2.

  17. Vaccination with EphA2-derived T cell-epitopes promotes immunity against both EphA2-expressing and EphA2-negative tumors

    Directory of Open Access Journals (Sweden)

    Hatano Manabu

    2004-11-01

    Full Text Available Abstract Background A novel tyrosine kinase receptor EphA2 is expressed at high levels in advanced and metastatic cancers. We examined whether vaccinations with synthetic mouse EphA2 (mEphA2-derived peptides that serve as T cell epitopes could induce protective and therapeutic anti-tumor immunity. Methods C57BL/6 mice received subcutaneous (s.c. vaccinations with bone marrow-derived dendritic cells (DCs pulsed with synthetic peptides recognized by CD8+ (mEphA2671–679, mEphA2682–689 and CD4+ (mEphA230–44 T cells. Splenocytes (SPCs were harvested from primed mice to assess the induction of cytotoxic T lymphocyte (CTL responses against syngeneic glioma, sarcoma and melanoma cell lines. The ability of these vaccines to prevent or treat tumor (s.c. injected MCA205 sarcoma or B16 melanoma; i.v. injected B16-BL6 establishment/progression was then assessed. Results Immunization of C57BL/6 mice with mEphA2-derived peptides induced specific CTL responses in SPCs. Vaccination with mEPhA2 peptides, but not control ovalbumin (OVA peptides, prevented the establishment or prevented the growth of EphA2+ or EphA2-negative syngeneic tumors in both s.c. and lung metastasis models. Conclusions These data indicate that mEphA2 can serve as an attractive target against which to direct anti-tumor immunity. The ability of mEphA2 vaccines to impact EphA2-negative tumors such as the B16 melanoma may suggest that such beneficial immunity may be directed against alternative EphA2+ target cells, such as the tumor-associated vascular endothelial cells.

  18. A next-generation cleaved, soluble HIV-1 Env trimer, BG505 SOSIP.664 gp140, expresses multiple epitopes for broadly neutralizing but not non-neutralizing antibodies.

    Directory of Open Access Journals (Sweden)

    Rogier W Sanders

    2013-09-01

    Full Text Available A desirable but as yet unachieved property of a human immunodeficiency virus type 1 (HIV-1 vaccine candidate is the ability to induce broadly neutralizing antibodies (bNAbs. One approach to the problem is to create trimeric mimics of the native envelope glycoprotein (Env spike that expose as many bNAb epitopes as possible, while occluding those for non-neutralizing antibodies (non-NAbs. Here, we describe the design and properties of soluble, cleaved SOSIP.664 gp140 trimers based on the subtype A transmitted/founder strain, BG505. These trimers are highly stable, more so even than the corresponding gp120 monomer, as judged by differential scanning calorimetry. They are also homogenous and closely resemble native virus spikes when visualized by negative stain electron microscopy (EM. We used several techniques, including ELISA and surface plasmon resonance (SPR, to determine the relationship between the ability of monoclonal antibodies (MAbs to bind the soluble trimers and neutralize the corresponding virus. In general, the concordance was excellent, in that virtually all bNAbs against multiple neutralizing epitopes on HIV-1 Env were highly reactive with the BG505 SOSIP.664 gp140 trimers, including quaternary epitopes (CH01, PG9, PG16 and PGT145. Conversely, non-NAbs to the CD4-binding site, CD4-induced epitopes or gp41ECTO did not react with the trimers, even when their epitopes were present on simpler forms of Env (e.g. gp120 monomers or dissociated gp41 subunits. Three non-neutralizing MAbs to V3 epitopes did, however, react strongly with the trimers but only by ELISA, and not at all by SPR and to only a limited extent by EM. These new soluble trimers are useful for structural studies and are being assessed for their performance as immunogens.

  19. Prediction and cloning linear Tcell epitopes of P14-3-3 antigen into pEGFP–N1 as a DNA vaccine model to induse immuno response against hydatidosis and it\\'s expression in CHO cell line

    Directory of Open Access Journals (Sweden)

    R mesri

    2015-11-01

    Full Text Available ABSTRACT Background & purpose: Hydatidosis is a zoonotic disease that caused by infection with the larvae of Echinococcus granulosus. Different antigens produced in larval stage of this parasite that recombinant vaccine base these antigens created significant immunity in infected animals. One of the important antigens is p14-3-3 that it's recombinant antigen created considerable immunity in mouse models. In this study according to the high immunity of antigen epitopes region the coding sequence of T-cell epitopes of P14-3-3 was cloned into pEGFP-N1vector in order to produce an effective DNA vaccine model to stimulate high level of Th1 immune response.   Material and method: In this study bioinformatics tools were used to prediction of linear T-Cell epitopes of Echinococcus granulosus P14-3-3 &zeta antigen. The nucleotide coding sequence of these epitopes was synthesized by PCR. the ampliqon was digested with XhoI restriction enzyme and cloned into pEGFP–N1 vector That has been purificated by modified sambrook method with CaCl2 and PEG6000..Positive colony was selected by direct colony PCR and confirmed by the sequencing.and evaluation of it's expression in Eukaryotic cells was done by transformed to CHO cell line with electroporation. Results: Linear T-cell epitopes of Echinococcus granulosus P14-3-3 after prediction,synthesis and amplification wae successfully cloned into pEGFP-N1 vector that purificated by new method with maximum vector and minimum RNA concentration.The expression of new constract in CHO cell line as a eukaryotic cells achivment by fluorescent microscope and will be used as a DNA vaccine model to evaluation immuno response in mouse models.   Discussion: Successfully cloning of The linear T-cell epitppes coding sequence of Echinococcus granulosus P14-3-3&zeta antigen into pEGFP-N1 verificated by sequencing and fluorscent microscope images demonstrated expression of recombinant protein in CHO cell line

  20. Relationship between the intrahepatic expression of 'e' and 'c' epitopes of the nucleocapsid protein of hepatitis B virus and viraemia.

    Science.gov (United States)

    Ballaré, M; Lavarini, C; Brunetto, M R; Petruzzelli, E; Dovis, M; Molino, G; Bonino, F

    1989-01-01

    The relationship between hepatitis B viraemia and intrahepatic HBV nucleocapsid proteins (HBcAg and HBeAg) was studied in 18 patients with chronic hepatitis B. Monoclonal antibodies (MoABs) were obtained in BALB/c mice primed with recombinant HBV nucleocapsid proteins. Four MoABs reacting with recombinant proteins gave positive results in competitive assays. Two reacted as anti-HBc and two as anti-HBe. One of them showed a strong affinity for the cytoplasmic, membrane-bound antigen (P23e) of infected hepatocytes while the latter showed a higher specificity for serum HBeAg than for the intrahepatic antigen. Anti-HBc MoABs had a staining capacity for liver cell nuclei comparable with that of polyclonal antibodies. Overall the anti-HBc MoABs stained the liver cell nuclei in 86% of cases, while anti-HBe MoABs stained in 58% of cases. The hepatocyte cytoplasm was stained by anti-HBc MoABs and anti-HBe MoABs in 64% and 72% of cases respectively. Not one of 12 control liver biopsies was stained. Viraemia (HBV-DNA) was measured by dot blot hybridization and was correlated with the number of hepatocytes containing the nucleocapsid antigen. The highest levels of HBV-DNA (greater than 10(8) genomes/ml) were detected in patients with prevalent nuclear staining while the lowest ones were observed in those with prevalent cytoplasmic expression of this antigen. The application of anti-HBV-nucleocapsid MoABs in diagnostics requires careful scrutiny since some are specific for the circulating antigen while others show a higher affinity for the intrahepatic antigen. PMID:2467769

  1. Structural features of N-glycans linked to glycoproteins expressed in three kinds of water plants: Predominant occurrence of the plant complex type N-glycans bearing Lewis a epitope.

    Science.gov (United States)

    Maeda, Megumi; Tani, Misato; Yoshiie, Takeo; Vavricka, Christopher J; Kimura, Yoshinobu

    2016-11-29

    The Japanese cedar pollen allergen (Cry j1) and the mountain cedar pollen allergen (Jun a1) are glycosylated with plant complex type N-glycans bearing Lewis a epitope(s) (Galβ1-3[Fucα1-4]GlcNAc-). The biological significance of Lewis a type plant N-glycans and their effects on the human immune system remain to be elucidated. Since a substantial amount of such plant specific N-glycans are required to evaluate immunological activity, we have searched for good plant-glycan sources to characterize Lewis a epitope-containing plant N-glycans. In this study, we have found that three water plants, Elodea nuttallii, Egeria densa, and Ceratophyllum demersum, produce glycoproteins bearing Lewis a units. Structural analysis of the N-glycans revealed that almost all glycoproteins expressed in these three water plants predominantly carry plant complex type N-glycans including the Lewis a type, suggesting that these water plants are good sources for preparation of Lewis a type plant N-glycans in substantial amounts.

  2. Electrophysiological properties of mouse and epitope-tagged human cardiac sodium channel Nav1.5 expressed in HEK293 cells [v2; ref status: indexed, http://f1000r.es/10d

    Directory of Open Access Journals (Sweden)

    Katja Reinhard

    2013-04-01

    Full Text Available Background: The pore-forming subunit of the cardiac sodium channel, Nav1.5, has been previously found to be mutated in genetically determined arrhythmias. Nav1.5 associates with many proteins that regulate its function and cellular localisation. In order to identify more in situ Nav1.5 interacting proteins, genetically-modified mice with a high-affinity epitope in the sequence of Nav1.5 can be generated. Methods: In this short study, we (1 compared the biophysical properties of the sodium current (INa generated by the mouse Nav1.5 (mNav1.5 and human Nav1.5 (hNav1.5 constructs that were expressed in HEK293 cells, and (2 investigated the possible alterations of the biophysical properties of the human Nav1.5 construct that was modified with specific epitopes. Results: The biophysical properties of mNav1.5 were similar to the human homolog. Addition of epitopes either up-stream of the N-terminus of hNav1.5 or in the extracellular loop between the S5 and S6 transmembrane segments of domain 1, significantly decreased the amount of INa and slightly altered its biophysical properties. Adding green fluorescent protein (GFP to the N-terminus did not modify any of the measured biophysical properties of hNav1.5. Conclusions: These findings have to be taken into account when planning to generate genetically-modified mouse models that harbour specific epitopes in the gene encoding mNav1.5.

  3. Identification of Autoantigen Epitopes in Alopecia Areata.

    Science.gov (United States)

    Wang, Eddy H C; Yu, Mei; Breitkopf, Trisia; Akhoundsadegh, Noushin; Wang, Xiaojie; Shi, Feng-Tao; Leung, Gigi; Dutz, Jan P; Shapiro, Jerry; McElwee, Kevin J

    2016-08-01

    Alopecia areata (AA) is believed to be a cell-mediated autoimmune hair loss disease. Both CD4 and cytotoxic CD8 T cells (CTLs) are important for the onset and progression of AA. Hair follicle (HF) keratinocyte and/or melanocyte antigen epitopes are suspected potential targets of autoreactive CTLs, but the specific epitopes have not yet been identified. We investigated the potential for a panel of known epitopes, expressed by HF keratinocytes and melanocytes, to induce activation of CTL populations in peripheral blood mononuclear cells. Specific synthetic epitopes derived from HF antigens trichohyalin and tyrosinase-related protein-2 induced significantly higher frequencies of response in AA CTLs compared with healthy controls (IFN-gamma secretion). Apoptosis assays revealed conditioned media from AA peripheral blood mononuclear cells stimulated with trichohyalin peptides elevated the expression of apoptosis markers in primary HF keratinocytes. A cytokine array revealed higher expression of IL-13 and chemokine ligand 5 (CCL5, RANTES) from AA peripheral blood mononuclear cells stimulated with trichohyalin peptides compared with controls. The data indicate that AA affected subjects present with an increased frequency of CTLs responsive to epitopes originating from keratinocytes and melanocytes; the activated CTLs secreted soluble factors that induced apoptosis in HF keratinocytes. Potentially, CTL response to self-antigen epitopes, particularly trichohyalin epitopes, could be a prognostic marker for human AA.

  4. Identification of immunodominant epitopes in Trypanosoma cruzi trypomastigote surface antigen-1 protein that mask protective epitopes.

    Science.gov (United States)

    Wrightsman, R A; Dawson, B D; Fouts, D L; Manning, J E

    1994-10-01

    The gene that encodes trypomastigote surface Ag-1 (TSA-1), a major surface Ag of the bloodstream trypomastigote stage of Trypanosoma cruzi, was expressed in a baculovirus expression system. To determine the epitope(s) in TSA-1 that was recognized during T. cruzi infection and after immunization with TSA-1, subregions of the TSA-1 gene were expressed in a bacterial expression system. As seen by Western blotting, both mice and rabbits immunized with recombinant TSA-1 protein, as well as T. cruzi-infected mice, developed strong immune responses to the carboxyl-proximal region of TSA-1, but show no reaction to the amino-proximal portion of TSA-1. When mice were immunized with either recombinant TSA-1 protein or the carboxyl-proximal region of TSA-1, they did not survive challenge with 10(3) bloodstream trypomastigotes. However, 70% of the mice immunized with the amino-proximal portion of TSA-1 survived challenge with 10(3) bloodstream trypomastigotes. Thus, the immune responses elicited by recombinant TSA-1 or the carboxyl-proximal portion of TSA-1 are nonprotective during T. cruzi infection. In contrast, vaccination with the amino proximal region of TSA-1 elicits a protective immune response. These results suggest that responses to immunodominant epitope(s) within the carboxyl-proximal portion of TSA-1 mask epitopes within the amino-proximal portion that are capable of stimulating host-protective immune responses. It is suggested that immunodominant regions in surface molecules such as TSA-1 may provide a mechanism for the parasite to evade the host immune response by directing the response away from epitopes that have the potential to elicit a reaction that is damaging to the parasite.

  5. IGHV1-69-Encoded Antibodies Expressed in Chronic Lymphocytic Leukemia React with Malondialdehyde-Acetaldehyde Adduct, an Immunodominant Oxidation-Specific Epitope

    DEFF Research Database (Denmark)

    Que, Xuchu; Widhopf Ii, George F; Amir, Shahzada

    2013-01-01

    with little or no somatic mutation, and restricted IGHD and IGHJ gene usage. We found that antibodies encoded by one particular IGHV1-69 subset, designated CLL69C, with the HCDR3 encoded by the IGHD3-3 gene in reading frame 2 and IGHJ6, specifically bound to oxidation-specific epitopes (OSE), which...... specifically with MAA-specific peptide mimotopes. Light chain shuffling indicated that non-stochastically paired L chain of IGLV3-9 contributes to the antigen binding of CLL69C. A nearly identical CLL69C Ig heavy chain was identified from an MAA-enriched umbilical cord phage displayed Fab library...

  6. Epitope prediction methods

    DEFF Research Database (Denmark)

    Karosiene, Edita

    Major histocompatibility complex (MHC) molecules play a crucial role in adaptive immunity by sampling peptides from self and non-self proteins to be recognised by the immune system. MHC molecules present peptides on cell surfaces for recognition by CD8+ and CD4+ T lymphocytes that can initiate...... immune responses. Therefore, it is of great importance to be able to identify peptides that bind to MHC molecules, in order to understand the nature of immune responses and discover T cell epitopes useful for designing new vaccines and immunotherapies. MHC molecules in humans, referred to as human...... on machine learning techniques. Several MHC class I binding prediction algorithms have been developed and due to their high accuracy they are used by many immunologists to facilitate the conventional experimental process of epitope discovery. However, the accuracy of these methods depends on data defining...

  7. Characterization of a cashew allergen, 11S globulin (Ana o 2), conformational epitope.

    Science.gov (United States)

    Robotham, Jason M; Xia, Lixin; Willison, LeAnna N; Teuber, Suzanne S; Sathe, Shridhar K; Roux, Kenneth H

    2010-05-01

    Both linear and conformational epitopes likely contribute to the allergenicity of tree nut allergens, yet, due largely to technical issues, few conformational epitopes have been characterized. Using the well studied recombinant cashew allergen, Ana o 2, an 11S globulin or legumin, we identified a murine monoclonal antibody which recognizes a conformational epitope and competes with patient IgE Ana o 2-reactive antibodies. This epitope is expressed on the large subunit of Ana o 2, but only when associated with an 11S globulin small subunit. Both Ana o 2 and the homologous soybean Gly m 6 small subunits can foster epitope expression, even when the natural N-terminal to C-terminal subunit order is reversed in chimeric molecules. The epitope, which is also expressed on native Ana o 2, is readily susceptible to destruction by physical and chemical denaturants.

  8. Construction and immunogenicity prediction of Plasmodium falciparum CTL epitope minigene vaccine

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    The minigenes encoding Plasmodium falciparum CTL epitopesrestricted to human MHC class I molecular HLA-A2 and HLA-B51, which were both at high frequency among Chinese population, were constructed as mono-epitope CTL vaccines named pcDNA3.1/tr and pcDNA3.1/ sh. The minigenes of the two epitopes were then tandem linked to form a dimeric CTL epitope minigene recombinant vaccine. After DNA transfection, the epitope minigenes were expressed respectively in two human cell lines, each bearing one MHC class I molecule named CIR/HLA-A2.1 and K562/HLA-B51. The intracellular expression of the CTL epitope minigenes not only enhanced the stability of HLA-A2.1 and HLA-B51 molecules but also increased the assemblage of MHC class I molecules on cell surfaces, which testified the specific process and presentation of those endogenous expressed epitopes. For the cells transfected with the dimeric minigene encoding two tandem linked epitopes, the expression and presentation of each epitope were also detected on cell membranes that bore different MHC class I molecules. It meant that the adjacency of the two CTL epitopes did not interfere with the specific process and presentation of each epitope. Compared with the ordinary CTL studies that inoculated synthesized epitope peptides with peripheral blood cells, this work aimed to process the epitopes directly inside HLA class I allele specific human cells, and thus theoretically imitated the same procedure in vivo. It was also an economical way to predict the immunogenicity of CTL epitopes at an early stage especially in laboratories with limited financial resource.

  9. Rhizobium rubi(T): a gram-negative phytopathogenic bacterium expressing the Lewis B epitope on the outer core of its lipooligosaccharide fraction.

    Science.gov (United States)

    Gargiulo, Valentina; Garozzo, Domenico; Lanzetta, Rosa; Molinaro, Antonio; Sturiale, Luisa; De Castro, Cristina; Parrilli, Michelangelo

    2008-07-21

    The structure of the core oligosaccharide from the phytopathogenic bacterium Rhizobium rubi was deduced by combining information from complementary chemical approaches (alkaline and acid hydrolysis), similar to the "overlap peptide" strategy. This structure is new and it contains two main oligosaccharide backbones that differ in the substitution degree of the external Kdo unit. The relevant feature shared by both oligosaccharides is the presence of a tetrasaccharide motif that is similar to the blood group Lewis B antigen (Le(B)). This epitope differs from Le(B) in the glycosidic configuration of the glucosamine unit (alpha and not beta) and in the occurrence of acetyls substituents at O3 and/or O4 of the galactose moiety. Other notable structural features are the location of the Dha residue, the presence of a alpha-glucose unit that is linked to the inner Kdo unit, the high number of acid sugars and the highly branched core structure.

  10. 猕猴B病毒gB蛋白特异性抗原表位的合成和表达%Synthesis and expression of monkey B virus glycoprotein B specific antigen epitope

    Institute of Scientific and Technical Information of China (English)

    隋丽华; 刘一; 赵彦斌; 张小飞; 崔晓霞; 叶华虎; 白杰英; 许琴; 孙兆增

    2013-01-01

    目的 获得可以用于B病毒检测的gB蛋白特异性表位重组抗原.方法 利用基因合成的方法,合成包含B病毒gB蛋白主要特性抗原表位的基因,通过定向克隆的方法,将该基因克隆到原核表达载体pET-22b中,构建了重组表达质粒.筛选出阳性重组质粒,转化到BL21感受态细胞,利用IPTG进行诱导表达.结果 成功的获得了B病毒gB蛋白的特异性抗原蛋白,该蛋白以可溶的形式表达.结论 利用原核表达系统,可以产生B病毒gB蛋白特异性表位重组抗原,可以作为B病毒的检测抗原.%Objective To get recombinant antigen including the antigen epitope of gB protein for the detection of B virus. Methods The gene fragment of the antigen epitope sequences of gB protein was synthesized and directly cloned into the pET-22b vector. The positive recombinant vector was transformed into BL21 competent cells and induced with IPTG. Results The synthesis gene fragment was expressed successfully in the Prokaryotic cells on the soluble form. Conclusion Prokaryotic expression system can produce soluble B virus gB protein antigen and can be used as the detection of B virus antigen

  11. Prokaryotic expression of MPSP double-epitope gene of bovine Theileria sergenti%牛瑟氏泰勒虫MPSP双表位基因的克隆及原核表达

    Institute of Scientific and Technical Information of China (English)

    钱年超; 贾立军; 薛书江; 张影; 张守发

    2011-01-01

    To express two major piroplasm surface protein (MPSP) epitopes of Theileria sergenti, two pairs of primers were designed and synthesized according to the gene sequence of T. Sergenti in GenBank. T. Sergenti genomic DNA was used as the template and 361 bp double-epitope fusion gene was amplified by SOE-PCR which was connected by linker (Gly4Ser)3. The fragment was cloned into pGEX-4T-2 to construct pGEX-4T-El-2. The recombinant protein was expressed in E. Coli BL21 inducted by LPTG, SDS-PAGE analysis showed that recombinant protein was about 39 ku, which was recognized by positive serum of T. Sergenti.%为了探究表位疫苗是否可以应用于预防牛瑟氏泰勒虫(Theileria sergenti),根据MPSP (Major piroplasm surface protein)基因序列(FJ515689.1)设计合成了2对引物,以T.sergenti基因组DNA为模板,通过SOE-PCR扩增出长约361 bp的双表位基因融合片段,2个表位基因通过linker (Gly4Ser)3相连.将该片段连接pGEX-4T-2,构建pGEX-4T-E1-2重组表达载体,转化BL21,IPTG诱导表达,经SDS-PAGE电泳显示表达了约39 ku的融合蛋白.Westem blot分析表明该双表位重组蛋白可与T.sergenti阳性血清发生特异性反应,表明融合蛋白具有反应原性.

  12. Immune epitope database analysis resource

    DEFF Research Database (Denmark)

    Kim, Yohan; Ponomarenko, Julia; Zhu, Zhanyang

    2012-01-01

    The immune epitope database analysis resource (IEDB-AR: http://tools.iedb.org) is a collection of tools for prediction and analysis of molecular targets of T- and B-cell immune responses (i.e. epitopes). Since its last publication in the NAR webserver issue in 2008, a new generation of peptide...... and can also be downloaded as software packages....

  13. Cloning and Expression of Epitopes of gD Protein of Pseudorabies Virus%猪伪狂犬病毒gD蛋白抗原表位的克隆及表达

    Institute of Scientific and Technical Information of China (English)

    刘芳; 张冲; 王寅彪; 赵朴; 邓瑞广; 李学伍

    2016-01-01

    According to the pseudorabies virus strain BJ/YT gD gene sequences in GenBank,a pair of primers were designed. The B-cell epitopes of gD was amplified,and the length of PCR product was 702 bp. The amplified fragment was cloned into the cloning vector pMD19-T successfully named pMD19-T-gD. The fragment was digested by BamH Ⅰ and Hind Ⅲ restriction enzymes and then cloned into the expression vector pET-28a named pET-28a-gD. The expression of protein was induced with IPTG,specific protein bands appeared at the site of 25 ku by SDS-PAGE electrophoresis. Western blot analysis showed that the expression product could be identified by pseudorabies virus positive serum. In this study, gD protein epitope was cloned and expressed successfully, and the expression product had good biological activity.%依据GenBank中猪伪狂犬病毒(PRV)BJ/YT株gD基因设计引物,扩增gD蛋白抗原表位,其设计扩增长度为702 bp,将扩增片段克隆到pMD19-T载体获得pMD19-T-gD.利用BamHⅠ和HindⅢ限制性内切酶双酶切pMD19-T-gD质粒,进行琼脂糖凝胶电泳并回收酶切片段,将回收片段与原核表达载体pET-28 a连接,成功构建了pET-28 a-gD表达载体.将表达载体转化感受态细胞Rosetta,IPTG诱导表达后,进行SDS–PAGE电泳,结果显示,在25 ku处出现特异性蛋白质条带.Western blot分析结果表明,表达产物能够被PRV的阳性血清识别.综上,成功克隆并表达了gD蛋白抗原表位,且其表达产物具有较好的生物学活性.

  14. Aberrant splicing in transgenes containing introns, exons, and V5 epitopes: lessons from developing an FSHD mouse model expressing a D4Z4 repeat with flanking genomic sequences.

    Directory of Open Access Journals (Sweden)

    Eugénie Ansseau

    Full Text Available The DUX4 gene, encoded within D4Z4 repeats on human chromosome 4q35, has recently emerged as a key factor in the pathogenic mechanisms underlying Facioscapulohumeral muscular dystrophy (FSHD. This recognition prompted development of animal models expressing the DUX4 open reading frame (ORF alone or embedded within D4Z4 repeats. In the first published model, we used adeno-associated viral vectors (AAV and strong viral control elements (CMV promoter, SV40 poly A to demonstrate that the DUX4 cDNA caused dose-dependent toxicity in mouse muscles. As a follow-up, we designed a second generation of DUX4-expressing AAV vectors to more faithfully genocopy the FSHD-permissive D4Z4 repeat region located at 4q35. This new vector (called AAV.D4Z4.V5.pLAM contained the D4Z4/DUX4 promoter region, a V5 epitope-tagged DUX4 ORF, and the natural 3' untranslated region (pLAM harboring two small introns, DUX4 exons 2 and 3, and the non-canonical poly A signal required for stabilizing DUX4 mRNA in FSHD. AAV.D4Z4.V5.pLAM failed to recapitulate the robust pathology of our first generation vectors following delivery to mouse muscle. We found that the DUX4.V5 junction sequence created an unexpected splice donor in the pre-mRNA that was preferentially utilized to remove the V5 coding sequence and DUX4 stop codon, yielding non-functional DUX4 protein with 55 additional residues on its carboxyl-terminus. Importantly, we further found that aberrant splicing could occur in any expression construct containing a functional splice acceptor and sequences resembling minimal splice donors. Our findings represent an interesting case study with respect to AAV.D4Z4.V5.pLAM, but more broadly serve as a note of caution for designing constructs containing V5 epitope tags and/or transgenes with downstream introns and exons.

  15. Galactosylated fucose epitopes in nematodes: increased expression in a Caenorhabditis mutant associated with altered lectin sensitivity and occurrence in parasitic species.

    Science.gov (United States)

    Yan, Shi; Bleuler-Martinez, Silvia; Plaza, David Fernando; Künzler, Markus; Aebi, Markus; Joachim, Anja; Razzazi-Fazeli, Ebrahim; Jantsch, Verena; Geyer, Rudolf; Wilson, Iain B H; Paschinger, Katharina

    2012-08-17

    The modification of α1,6-linked fucose residues attached to the proximal (reducing-terminal) core N-acetylglucosamine residue of N-glycans by β1,4-linked galactose ("GalFuc" epitope) is a feature of a number of invertebrate species including the model nematode Caenorhabditis elegans. A pre-requisite for both core α1,6-fucosylation and β1,4-galactosylation is the presence of a nonreducing terminal N-acetylglucosamine; however, this residue is normally absent from the final glycan structure in invertebrates due to the action of specific hexosaminidases. Previously, we have identified two hexosaminidases (HEX-2 and HEX-3) in C. elegans, which process N-glycans. In the present study, we have prepared a hex-2;hex-3 double mutant, which possesses a radically altered N-glycomic profile. Whereas in the double mutant core α1,3-fucosylation of the proximal N-acetylglucosamine was abolished, the degree of galactosylation of core α1,6-fucose increased, and a novel Galα1,2Fucα1,3 moiety attached to the distal core N-acetylglucosamine residue was detected. Both galactosylated fucose moieties were also found in two parasitic nematodes, Ascaris suum and Oesophagostomum dentatum. As core modifications of N-glycans are known targets for fungal nematotoxic lectins, the sensitivity of the C. elegans double hexosaminidase mutant was assessed. Although this mutant displayed hypersensitivity to the GalFuc-binding lectin CGL2 and the N-acetylglucosamine-binding lectin XCL, the mutant was resistant to CCL2, which binds core α1,3-fucose. Thus, the use of C. elegans mutants aids the identification of novel N-glycan modifications and the definition of in vivo specificities of nematotoxic lectins with potential as anthelmintic agents.

  16. The immunodominant HLA-A2-restricted MART-1 epitope is not presented on the surface of many melanoma cell lines

    DEFF Research Database (Denmark)

    Sørensen, Rikke Baek; Junker, Niels; Kirkin, Alexei;

    2009-01-01

    of the melanoma cells with the MART-1 epitope. Thus, the very frequently used MART-1 epitope was not expressed on the surface of many melanoma cell lines. Our data emphasize that the selected tumor antigens and/or epitopes are critical for the outcome of anti-cancer immunotherapy....

  17. An epitope delivery system for use with recombinant mycobacteria

    NARCIS (Netherlands)

    Hetzel, C.; Janssen, R.; Ely, S.J.; Kristensen, N.M.; Bunting, K.; Cooper, J.B.; Lamb, J.R.; Young, D.B.; Thole, J.E.R.

    1998-01-01

    We have developed a novel epitope delivery system based on the insertion of peptides within a permissive loop of a bacterial superoxide dismutase molecule. This system allowed high-level expression of heterologous peptides in two mycobacterial vaccine strains, Mycobacterium bovis bacille Calmette- G

  18. Expression and use of a multi-epitope antigen derived from the G2 glycoprotein of Hantavirus%汉坦病毒G2糖蛋白多表位抗原的表达及应用

    Institute of Scientific and Technical Information of China (English)

    杨东靖; 陈锦英; 苏旭; 李力; 吕莉琨

    2013-01-01

    目的 对汉坦病毒G2糖蛋白多表位抗原(multi-epitope antigen,MEA)进行原核表达和纯化,建立MEA-ELISA方法并用于肾综合征出血热(HFRS)患者血清特异性抗体的检测. 方法 利用pET原核表达系统表达MEA,并进行镍亲和纯化;利用Western blot方法对MEA进行免疫反应性鉴定,并建立MEA-ELISA方法,用于检测HFRS患者血清特异性抗体. 结果 原核表达系统E coli BL21/pET32a-mea能表达分子质量单位为36.9 ku的MEA蛋白,纯化后的蛋白浓度为0.15 mg/ml,纯度>95%,Western blot显示目的蛋白能被HFRS患者血清识别.用建立的MEA-ELISA方法检测120例HFRS患者血清的阳性率为90.83%,检测60例健康人血清全部为阴性. 结论 重组表达蛋白MEA具有良好的免疫反应性,建立的MEA-ELISA方法可用于HFRS患者血清特异性抗体的检测.%Objectives To express and purify a multi-epitope antigen (MEA) derived from the G2 glycoprotein of Hantavirus and establish a method of MEA-ELISA to detect specific antibodies in serum from patients with hemorrhagic fever with renal syndrome (HFRS).Methods MEA was expressed with a pET prokaryotic system and purified with nickel affinity chromatography.The immunoreaction specificity of MEA was investigated using Western blotting.A method of MEA-ELISA was established to detect specific antibodies in patients with HFRS.Results An MEA was successfully expressed with a pET prokaryotic expression system.The MEA protein had a molecular weight of 36.9 ku,and the yield after purification was 0.15 mg/ml.Western blot analysis indicated the target band of MEA was recognized by serum from patients with HFRS.MEA-ELISA results were positive for 120 patients with HFRS at a rate of 90.83% and were negative for 60 healthy individuals.Conclusion The recombinant protein MEA had good immunoreactive specificity.MEA ELISA was able to detect specific antibodies in serum from patients with HFRS.

  19. A rapid and safe plasmid isolation method for efficient engineering of recombinant lactobacilli expressing immunogenic or tolerogenic epitopes for oral administration

    NARCIS (Netherlands)

    Maassen, C.B.M.

    1999-01-01

    Recombinant lactobacilli are being developed which can be used as expression and delivery vectors of heterologous antigens in oral vaccination and other therapeutic applications. Because most Lactobacillus strains do not accept ligation mixtures, sufficiently pure plasmid DNA needs to be isolated fr

  20. Identification of a variant antigenic neutralizing epitope in hypervariable region 1 of avian leukosis virus subgroup J.

    Science.gov (United States)

    Hou, Minbo; Zhou, Defang; Li, Gen; Guo, Huijun; Liu, Jianzhu; Wang, Guihua; Zheng, Qiankun; Cheng, Ziqiang

    2016-03-08

    Avian leukosis virus subgroup J (ALV-J) is a hypervariable oncogenic retrovirus that causes great economic loss in poultry. Antigenic variations in the variable regions make the development of an effective vaccine a challenging task. In the present study, we identified a variant antigenic neutralizing epitope using reverse vaccinology methods. First, we predicted the B-cell epitopes in gp85 gene of ALV-J strains by DNAman and bioinformatics. Fourteen candidate epitopes were selected and linked in tandem with glycines or serines as a multi-epitope gene. The expressed protein of multi-epitope gene can induce high-titer antibody that can recognize nature ALV-J and neutralize the infectivity of ALV-J strains. Next, we identified a high effective epitope using eight overlapping fragments of gp85 gene reacting with mAb 2D5 and anti-multi-epitope sera. The identified epitope contained one of the predicted epitopes and localized in hyervariable region 1 (hr1), indicating a variant epitope. To better understand if the variants of the epitope have a good antigenicity, we synthesized four variants to react with mAb 2D5 and anti-ALV-J sera. The result showed that all variants could react with the two kinds of antibodies though they showed different antigenicity, while could not react with ALV-J negative sera. Thus, the variant antigenic neutralizing epitope was determined as 137-LRDFIA/E/TKWKS/GDDL/HLIRPYVNQS-158. The result shows a potential use of this variant epitopes as a novel multi-epitope vaccine against ALV-J in poultry.

  1. 以腺病毒为载体表达猪α(1,3)半乳糖基转移酶 反义RNA抑制Galα(1,3)Gal抗原表位的表达%Adenovirus-mediated expression of antisense RNA transcripts complementary to pig a(1,3) galactosyltransferase mRNA inhibits expression of Gal α(1,3) Gal epitope

    Institute of Scientific and Technical Information of China (English)

    邢力; 夏国宏; 白旭芳; 费俭; 郭礼和

    2000-01-01

    目的:尝试以反义RNA的方法抑制Galα(1,3)Gal抗原表位(gal抗原)的表达.方法:以人腺病毒载体表达猪α(1,3)半乳糖基转移酶基因的反义RNA.流式细胞术比较H血型抗原和gal抗原的表达水平.结果:构建了表达反义RNA的重组腺病毒载体Ad5anti-sGT600和Adanti-sGTll00.反义RNA的表达使NIH3T3细胞表面的gal抗原表位下降约30%.另外,反义RNA与人分泌型α(1,2)岩藻糖基转移酶的共同作用可使gal抗原表位的水平进一步下降.结论:重组腺病毒Adanti-sGT600和Ad5anti-sGTll00可有效降低gal抗原表位的表达.%AIM: To examine the effects of the expression of antisense RNA transcripts complementary to the pig α(1,3) galactosyltransferase [α(1,3)GT]mRNA on the expression of Gal α(1,3) Gal structure (gal epitope) in cultured cell lines. METHODS: Human adenoviral vectors were used to mediate the expression of antisense RNA. The expression levels of H blood group antigens and gal epitopes were analyzed by flow cytometry using FITC-UEAI and FITC-GS-IB4 lectins, respectively. RESULTS: Recombinant adenoviruses, Ad5anti-sGT600 and Ad5anti-sGTll00, which express antisense RNA complementary to different regions of the pig α (1,3) GT mRNA, were constructed and used to infect cell line of NIH3T3. The results showed about 30% reduction in the expression level of gal epitopes on the surface of NIH3T3 cells. In addition, co-expression of human secretor type α(1,2) fucosyltransferase [α(1,2)Fr]cDNA and antisense RNA complementary to the pig α(1 ,3) GT mRNA led to a further reduction in the gal epitope level. CONCLUSION: Recombinant adenoviruses, Ad5anti-sGT600 and Ad5anti-sGTll00, are effective to down-regulate the gal epitope expression.

  2. Immune Epitope Database and Analysis Resource (IEDB)

    Data.gov (United States)

    U.S. Department of Health & Human Services — This repository contains antibody/B cell and T cell epitope information and epitope prediction and analysis tools for use by the research community worldwide. Immune...

  3. Expression and purification of an immunogenic dengue virus epitope using a synthetic consensus sequence of envelope domain III and Saccharomyces cerevisiae.

    Science.gov (United States)

    Nguyen, Ngoc-Luong; Kim, Jung-Mi; Park, Jin-Ah; Park, Seung-Moon; Jang, Yong-Suk; Yang, Moon-Sik; Kim, Dae-Hyuk

    2013-04-01

    A synthetic consensus gene was designed based on residues of the amino acid sequences of dengue envelope domain III (scEDIII) from all four serotypes, and codon optimization for expression was conducted using baker's yeast, Saccharomyces cerevisiae. The synthetic gene was cloned into a yeast episomal expression vector, pYEGPD-TER, which was designed to direct cloned gene expression using the glyceraldehyde-3-phosphate dehydrogenase (GPD) promoter, a functional signal peptide of the amylase 1A protein from rice, and the GAL7 terminator. PCR and back-transformation into Escherichia coli confirmed the presence of the scEDIII gene-containing plasmid in the transformants. Northern blot analysis showed the presence of the scEDIII-specific transcript. Western blot analysis indicated that expressed scEDIII, with mobility similar to purified EDIII from E. coli, was successfully secreted into the culture media. Quantitative ELISA revealed that the recombinant scEDIII comprised approximately 0.1-0.6% of cell-free extract. In addition, 0.1-0.6 mg of scEDIII protein per liter of culture filtrate was detected on day 1 and peaked on day 3 after cultivation. The secreted scEDIII protein can be purified to ≥90% purity with 85% recovery using a simple ion-exchange FPLC followed by molecular weight cut-off. Upon administration of the purified protein to mice, mouse sera contained antibodies that were specific to all four serotypes of dengue virus. Moreover, a balanced immune response against all four serotypes was observed, suggesting that it may be possible to develop an effective tetravalent dengue vaccine using S. cerevisiae.

  4. HIV-1包膜蛋白gp41优势抗原的构建及可溶性表达%Construction and Prokaryotic Expression of Soluble HIV-1 gp41 Dominant Epitopes

    Institute of Scientific and Technical Information of China (English)

    杜刚; 江蒙蒙; 尹林; 王宏萍; 周晓明

    2011-01-01

    目的:筛选1型人免疫缺陷病毒(HIV-1)中国流行株中包膜蛋白gp41的优势抗原片段,构建具有区域流行代表性的HIV-1 gp41重组抗原,为改进现有HIV-1初筛试剂盒中使用的同类抗原奠定基础.方法:利用免疫斑点杂交和生物信息学方法,从收集自重庆、广州、上海的区域代表性150份HIV-1感染者血清标本中筛选gp41抗原性强的候选样本,利用RT-PCR及巢式PCR方法扩增包含重要抗原表位决定蔟的gp41基因片段,与原核表达载体pQE30连接,转化大肠杆菌M15构建gp41重组抗原表达菌株,表达后经亲和层析纯化、SDS-PAGE和Western印迹鉴定.结果:兔源HRP标记的gp41多抗能识别标本中gp41抗原性差异,得到候选样本,扩增包含gp41主要抗原表位片段;构建了包含gp41抗原表达簇的重组原核表达质粒,表达、纯化后经His标签抗体Western印迹鉴定为阳性.结论:高纯度的重组优势gp41抗原的构建和鉴定,为进一步改进现有HIV初筛诊断奠定了基础.%Objective: To prepare the major antigen fragment of gp41 of HIV-1 based on main epidemic strains in China and to construct representative gp41 recombinant antigen of HIV-1 for further improving the efficacy of current HIV screening test. Methods: Combined with viral load of each sample, dot blot and bioinfomatic analysis were to identify two candidate samples from 150 HIV-1 postive serums collected from Chongqing, Guangzhou and Shanghai. Subsequently, RT-PCR and nested PCR were performed to synthesize gp41 epitope-specific sequences, then the cloned fragments containing dominant gp41 epitopes cotransformed with the backbone vector pQE30 into E.coil M15 was performed to obtain recombinant gp41 expression strain. Results: Rabbit HRP-labelled anti-gp41 polyclonal antibody could recognize the different gp41 intensity in 150 samples, from which the result led to the successful selection of two candidate samples. Then we constructed the recombinant

  5. Human Antibodies that Recognize Novel Immunodominant Quaternary Epitopes on the HIV-1 Env Protein

    Science.gov (United States)

    Hicar, Mark D.; Chen, Xuemin; Sulli, Chidananda; Barnes, Trevor; Goodman, Jason; Sojar, Hakimuddin; Briney, Bryan; Willis, Jordan; Chukwuma, Valentine U.; Kalams, Spyros A.; Doranz, Benjamin J.; Spearman, Paul; Crowe, James E.

    2016-01-01

    Numerous broadly neutralizing antibodies (Abs) target epitopes that are formed or enhanced during mature HIV envelope formation (i.e. quaternary epitopes). Generally, it is thought that Env epitopes that induce broadly neutralizing Abs are difficult to access and poorly immunogenic because of the characteristic oligomerization, conformational flexibility, sequence diversity and extensive glycosylation of Env protein. To enhance for isolation of quaternary epitope-targeting Abs (QtAbs), we previously used HIV virus-like particles (VLPs) to bind B cells from long-term non-progressor subjects to identify a panel of monoclonal Abs. When expressed as recombinant full-length Abs, a subset of these novel Abs exhibited the binding profiles of QtAbs, as they either failed to bind to monomeric Env protein or showed much higher affinity for Env trimers and VLPs. These QtAbs represented a significant proportion of the B-cell response identified with VLPs. The Ab genes of these clones were highly mutated, but they did not neutralize common HIV strains. We sought to further define the epitopes targeted by these QtAbs. Competition-binding and mapping studies revealed these Abs targeted four separate epitopes; they also failed to compete for binding by Abs to known major neutralizing epitopes. Detailed epitope mapping studies revealed that two of the four epitopes were located in the gp41 subunit of Env. These QtAbs bound pre-fusion forms of antigen and showed differential binding kinetics depending on whether oligomers were produced as recombinant gp140 trimers or as full-length Env incorporated into VLPs. Antigenic regions within gp41 present unexpectedly diverse structural epitopes, including these QtAb epitopes, which may be targeted by the naturally occurring Ab response to HIV infection. PMID:27411063

  6. Human Antibodies that Recognize Novel Immunodominant Quaternary Epitopes on the HIV-1 Env Protein.

    Directory of Open Access Journals (Sweden)

    Mark D Hicar

    Full Text Available Numerous broadly neutralizing antibodies (Abs target epitopes that are formed or enhanced during mature HIV envelope formation (i.e. quaternary epitopes. Generally, it is thought that Env epitopes that induce broadly neutralizing Abs are difficult to access and poorly immunogenic because of the characteristic oligomerization, conformational flexibility, sequence diversity and extensive glycosylation of Env protein. To enhance for isolation of quaternary epitope-targeting Abs (QtAbs, we previously used HIV virus-like particles (VLPs to bind B cells from long-term non-progressor subjects to identify a panel of monoclonal Abs. When expressed as recombinant full-length Abs, a subset of these novel Abs exhibited the binding profiles of QtAbs, as they either failed to bind to monomeric Env protein or showed much higher affinity for Env trimers and VLPs. These QtAbs represented a significant proportion of the B-cell response identified with VLPs. The Ab genes of these clones were highly mutated, but they did not neutralize common HIV strains. We sought to further define the epitopes targeted by these QtAbs. Competition-binding and mapping studies revealed these Abs targeted four separate epitopes; they also failed to compete for binding by Abs to known major neutralizing epitopes. Detailed epitope mapping studies revealed that two of the four epitopes were located in the gp41 subunit of Env. These QtAbs bound pre-fusion forms of antigen and showed differential binding kinetics depending on whether oligomers were produced as recombinant gp140 trimers or as full-length Env incorporated into VLPs. Antigenic regions within gp41 present unexpectedly diverse structural epitopes, including these QtAb epitopes, which may be targeted by the naturally occurring Ab response to HIV infection.

  7. Parallel immunizations of rabbits using the same antigen yield antibodies with similar, but not identical, epitopes.

    Directory of Open Access Journals (Sweden)

    Barbara Hjelm

    Full Text Available A problem for the generation of polyclonal antibodies is the potential difficulties for obtaining a renewable resource due to batch-to-batch variations when the same antigen is immunized into several separate animals. Here, we have investigated this issue by determining the epitopes of antibodies generated from parallel immunizations of rabbits with recombinant antigens corresponding to ten human protein targets. The epitopes were mapped by both a suspension bead array approach using overlapping synthetic 15-mer peptides and a bacterial display approach using expression of random fragments of the antigen on the surface of bacteria. Both methods determined antibody binding with the aid of fluorescent-based analysis. In addition, one polyclonal antibody was fractionated by peptide-specific affinity capture for in-depth comparison of epitopes. The results show that the same antigen immunized in several rabbits yields polyclonal antibodies with similar epitopes, but with larger differences in the relative amounts of antibodies to the different epitopes. In some cases, unique epitopes were observed for one of the immunizations. The results suggest that polyclonal antibodies generated by repeated immunizations do not display an identical epitope pattern, although many of the epitopes are similar.

  8. Predefined GPGRAFY-Epitope-Specific Monoclonal Antibodies with Different Activities for Recognizing Native HIV-1 gp120

    Institute of Scientific and Technical Information of China (English)

    蓝灿辉; 田海军; 陈应华

    2004-01-01

    A seven-amino acid epitope GPGRAFY at the tip of the V3 loop in HIV-1 gp120 is the principal neutralizing epitope,and a subset of anti-V3 antibodies specific for this epitope shows a broad range of neutralizing activity.GPGRAFY-epitope-specific neutralizing antibodies were produced using predefined GPGRAFY-epitope-specific peptides instead of a natural or recombinant gp120 bearing this epitope.All six monoclonal antibodies (mAbs) could recognize the GPGRAFY-epitope on peptides and two of the antibodies,9D8 and 2D7,could recognize recombinant gp120 in enzymelinked immunosorkentassy (ELISA) assays.In the flow cytometry analysis,the mAbs 9D8 and 2D7 could bind to HIV-Env+ CHO-WT cells and the specific bindings could be inhibited by the GPGRAFY-epitope peptide,which suggests that these two mAbs could recognize the native envelope protein gp120 expressed on the cell membrane.However,in syncytium assays,none of the mAbs was capable of inhibiting HIV-Env-mediated cell membrane fusion.The different activities for recognizing native HIV-1 gp120 might be associated with different antibody affinities against the epitopes.The development of conformational mimics of the neutralization epitope in the gp120 V3 loop could elicit neutralizing mAbs with high affinity.

  9. Construction and characterization of an HCV-derived multi-epitope peptide antigen containing B-cell HVR1 mimotopes and T-cell conserved epitopes

    Institute of Scientific and Technical Information of China (English)

    GAO; Jun; GONG; Yuping; ZHAO; Ping; ZHU; Qing; YANG; Xiaoping; QI; Zhongtian

    2006-01-01

    Hepatitis C (HCV) genome is highly variable, particularly in the hypervariable region 1(HVR1) of its E2 envelope gene. The variability of HCV genome has been a major obstacle for developing HCV vaccines. Due to B-cell HVR1 mimotopes mimicking the antigenicity of natural HVR1 epitopes and some T-cell epitopes from the consensus sequence of HCV genes conserving among the different HCV genotypes, we synthesized an minigene of HCV-derived multi-epitope peptide antigen (CMEP), which contains 9 B-cell HVR1 mimotopes in E2, 2 conserved CTL epitopes in C, 1conserved CTL epitope in NS3 and 1 conserved Th epitope in NS3. This minigene was cloned into a GST expression vector to generate a fusion protein GST-CMER The immunogenic properties of CEMP were characterized by HCV infected patients' sera, and found that the reactivity frequency reached 75%. The cross reactivity of anti-CEMP antibody with different natural HVR1 variants was up to 90%. Meanwhile, we constructed an HCV DNA vaccine candidate, plasmid pVAX1.0-st-CMEP carrying the recombinant gene (st) of a secretion signal peptide and PADRE universal Th cell epitope sequence in front of the CMEP minigene. Immunization of rabbits with pVAX1.0-st-CMEP resulted in the production of antibody, which was of the same cross reactivity as the fusion protein GST-CMEP.Our findings indicate that the HCV-derived multi-epitope peptide antigen in some degree possessed the characteristics of neutralizing HCV epitopes, and would be of the value as a candidate for the development of HCV vaccines.

  10. Breaking tolerance in transgenic mice expressing the human TSH receptor A-subunit: thyroiditis, epitope spreading and adjuvant as a 'double edged sword'.

    Directory of Open Access Journals (Sweden)

    Sandra M McLachlan

    Full Text Available Transgenic mice with the human thyrotropin-receptor (TSHR A-subunit targeted to the thyroid are tolerant of the transgene. In transgenics that express low A-subunit levels (Lo-expressors, regulatory T cell (Treg depletion using anti-CD25 before immunization with adenovirus encoding the A-subunit (A-sub-Ad breaks tolerance, inducing extensive thyroid lymphocytic infiltration, thyroid damage and antibody spreading to other thyroid proteins. In contrast, no thyroiditis develops in Hi-expressor transgenics or wild-type mice. Our present goal was to determine if thyroiditis could be induced in Hi-expressor transgenics using a more potent immunization protocol: Treg depletion, priming with Complete Freund's Adjuvant (CFA + A-subunit protein and further Treg depletions before two boosts with A-sub-Ad. As controls, anti-CD25 treated Hi- and Lo-expressors and wild-type mice were primed with CFA+ mouse thyroglobulin (Tg or CFA alone before A-sub-Ad boosting. Thyroiditis developed after CFA+A-subunit protein or Tg and A-sub-Ad boosting in Lo-expressor transgenics but Hi- expressors (and wild-type mice were resistant to thyroiditis induction. Importantly, in Lo-expressors, thyroiditis was associated with the development of antibodies to the mouse TSHR downstream of the A-subunit. Unexpectedly, we observed that the effect of bacterial products on the immune system is a "double-edged sword". On the one hand, priming with CFA (mycobacteria emulsified in oil plus A-subunit protein broke tolerance to the A-subunit in Hi-expressor transgenics leading to high TSHR antibody levels. On the other hand, prior treatment with CFA in the absence of A-subunit protein inhibited responses to subsequent immunization with A-sub-Ad. Consequently, adjuvant activity arising in vivo after bacterial infections combined with a protein autoantigen can break self-tolerance but in the absence of the autoantigen, adjuvant activity can inhibit the induction of immunity to autoantigens

  11. Breaking tolerance in transgenic mice expressing the human TSH receptor A-subunit: thyroiditis, epitope spreading and adjuvant as a 'double edged sword'.

    Science.gov (United States)

    McLachlan, Sandra M; Aliesky, Holly A; Chen, Chun-Rong; Chong, Gao; Rapoport, Basil

    2012-01-01

    Transgenic mice with the human thyrotropin-receptor (TSHR) A-subunit targeted to the thyroid are tolerant of the transgene. In transgenics that express low A-subunit levels (Lo-expressors), regulatory T cell (Treg) depletion using anti-CD25 before immunization with adenovirus encoding the A-subunit (A-sub-Ad) breaks tolerance, inducing extensive thyroid lymphocytic infiltration, thyroid damage and antibody spreading to other thyroid proteins. In contrast, no thyroiditis develops in Hi-expressor transgenics or wild-type mice. Our present goal was to determine if thyroiditis could be induced in Hi-expressor transgenics using a more potent immunization protocol: Treg depletion, priming with Complete Freund's Adjuvant (CFA) + A-subunit protein and further Treg depletions before two boosts with A-sub-Ad. As controls, anti-CD25 treated Hi- and Lo-expressors and wild-type mice were primed with CFA+ mouse thyroglobulin (Tg) or CFA alone before A-sub-Ad boosting. Thyroiditis developed after CFA+A-subunit protein or Tg and A-sub-Ad boosting in Lo-expressor transgenics but Hi- expressors (and wild-type mice) were resistant to thyroiditis induction. Importantly, in Lo-expressors, thyroiditis was associated with the development of antibodies to the mouse TSHR downstream of the A-subunit. Unexpectedly, we observed that the effect of bacterial products on the immune system is a "double-edged sword". On the one hand, priming with CFA (mycobacteria emulsified in oil) plus A-subunit protein broke tolerance to the A-subunit in Hi-expressor transgenics leading to high TSHR antibody levels. On the other hand, prior treatment with CFA in the absence of A-subunit protein inhibited responses to subsequent immunization with A-sub-Ad. Consequently, adjuvant activity arising in vivo after bacterial infections combined with a protein autoantigen can break self-tolerance but in the absence of the autoantigen, adjuvant activity can inhibit the induction of immunity to autoantigens (like the

  12. Mast Cells Produce a Unique Chondroitin Sulfate Epitope.

    Science.gov (United States)

    Farrugia, Brooke L; Whitelock, John M; O'Grady, Robert; Caterson, Bruce; Lord, Megan S

    2016-02-01

    The granules of mast cells contain a myriad of mediators that are stored and protected by the sulfated glycosaminoglycan (GAG) chains that decorate proteoglycans. Whereas heparin is the GAG predominantly associated with mast cells, mast cell proteoglycans are also decorated with heparan sulfate and chondroitin sulfate (CS). This study investigated a unique CS structure produced by mast cells that was detected with the antibody clone 2B6 in the absence of chondroitinase ABC digestion. Mast cells in rodent tissue sections were characterized using toluidine blue, Leder stain and the presence of mast cell tryptase. The novel CS epitope was identified in rodent tissue sections and localized to cells that were morphologically similar to cells chemically identified as mast cells. The rodent mast cell-like line RBL-2H3 was also shown to express the novel CS epitope. This epitope co-localized with multiple CS proteoglycans in both rodent tissue and RBL-2H3 cultured cells. These findings suggest that the novel CS epitope that decorates mast cell proteoglycans may play a role in the way these chains are structured in mast cells.

  13. 单纯疱疹病毒Ⅰ型的Th细胞和B细胞表位重组核酸疫苗的构建及真核表达%Construction and Eukaryotic Expression of HSV?1 Th and B Lymphocytes Recombinant Multi?epitope DNA Vaccine

    Institute of Scientific and Technical Information of China (English)

    刘贤洁; 孙原; 马小力

    2015-01-01

    Objective To construct the recombinant multi?epitope DNA vaccine of HSV?1 Th and B lymphocytes of gB?gD protein,and to identify its eukaryotic expression. Methods The epitopes of HSV?1 gB?gD protein were analyzed by bioinformatics analysis software. HSV?1 Th and B lym?phocyte recombinant multi?epitope gene(Th/B gene)was designed and synthesized. Th/B gene was cloned into pVAX1 to synthesize pVAX1?Th/B. pVAX1?Th/B was then transfected into COS?7 cell,and the protein expression was identified by Western blot. Results The epitopes of Th and B lymphocytes of HSV?1 gB and gD were predicted,and the eukaryotic expression plasmid pf pVAX1?Th/B was successfully constructed and con?firmed by sequencing. In addition,the in vivo protein expression of the recombinant DNA was confirmed by Western blot. Conclusion HSV?1 Th and B lymphocyte recombinant multi?epitope DNA vaccine was successfully synthesized in this study. HSV?1 Th and B lymphocyte recombinant multi?epitope DNA vaccine can be successfully expressed in isolated eukaryotic cells. This study provides a feasible solution for developing DNA vac?cine against HSV?1 infection.%目的 构建单纯疱疹病毒Ⅰ型(HSV?1)糖蛋白B(gB)和糖蛋白D(gD)的Th细胞和B细胞表位重组串联核酸疫苗,并鉴定其真核表达.方法 利用生物信息学分析软件预测HSV?1 gB和gD的Th细胞表位和B细胞表位,结合已发表文献,设计合成HSV?1的Th细胞和B细胞表位重组串联抗原基因(Th/B基因).将Th/B基因定向克隆入真核表达载体pVAX1,构建真核表达质粒pVAX1?Th/B.用制备的质粒转染COS?7细胞,裂解细胞后经Western blot鉴定其蛋白表达.结果 预测得到了HSV?1 gB和gD的Th细胞和B细胞表位,成功构建真核表达质粒pVAX1?Th/B,经测序确认序列正确.SDS?PAGE分离出相应大小的蛋白表达片段,经Western blot鉴定并证实该重组核酸疫苗能够在体外真核细胞中表达.结论 成功构建HSV?1 Th细胞和B细胞表位重组串联核酸

  14. The Immune Epitope Database 2.0

    DEFF Research Database (Denmark)

    Hoof, Ilka; Vita, R; Zarebski, L;

    2010-01-01

    The Immune Epitope Database (IEDB, www.iedb.org) provides a catalog of experimentally characterized B and T cell epitopes, as well as data on Major Histocompatibility Complex (MHC) binding and MHC ligand elution experiments. The database represents the molecular structures recognized by adaptive...... immune receptors and the experimental contexts in which these molecules were determined to be immune epitopes. Epitopes recognized in humans, nonhuman primates, rodents, pigs, cats and all other tested species are included. Both positive and negative experimental results are captured. Over the course...

  15. Mapping of a conformational epitope on the cashew allergen Ana o 2: a discontinuous large subunit epitope dependent upon homologous or heterologous small subunit association.

    Science.gov (United States)

    Xia, Lixin; Willison, LeAnna N; Porter, Lauren; Robotham, Jason M; Teuber, Suzanne S; Sathe, Shridhar K; Roux, Kenneth H

    2010-05-01

    The 11S globulins are members of the cupin protein superfamily and represent an important class of tree nut allergens for which a number of linear epitopes have been mapped. However, specific conformational epitopes for these allergens have yet to be described. We have recently reported a cashew Ana o 2 conformational epitope defined by murine mAb 2B5 and competitively inhibited by a subset of patient IgE antibodies. The 2B5 epitope appears to reside on the large (acidic) subunit, is dependent upon small (basic) subunit association for expression, and is highly susceptible to denaturation. Here we fine map the epitope using a combination of recombinant chimeric cashew Ana o 2-soybean Gly m 6 chimeras, deletion and point mutations, molecular modeling, and electron microscopy of 2B5-Ana o 2 immune complexes. Key residues appear confined to a 24 amino acid segment near the N-terminus of the large subunit peptide, a portion of which makes direct contact with the small subunit. These data provide an explanation for both the small subunit dependence and the structurally labile nature of the epitope.

  16. The epitopes in wheat proteins for defining toxic units relevant to human health.

    Science.gov (United States)

    Juhász, Angéla; Gell, Gyöngyvér; Békés, Frank; Balázs, Ervin

    2012-11-01

    Wheat-related disorders are well-studied health problems. Knowledge of the composition and amounts of epitopes present in a single wheat sample represents a significant gap, and the detailed wheat proteome datasets now available can provide the necessary information to carry out an estimation of allergen prediction for a single cultivar. The combined use of genome sequence and allergen databases, prediction methodology, and cereal chemistry results in better understanding of the level of toxicity present in the end-products produced from wheat flour. The workflow presented in this review provides information about the number and distribution of epitopes at single protein, or protein fraction, levels. In addition, epitopes present in the highest frequency and harmful proteins expressed in the highest amount can be identified. The "epitope toxicity" value obtained in this way is a significant research output from the analysis of large datasets that can be applied to the food industry.

  17. Induction of multi-epitope specific antibodies against HIV-1 by multi-epitope vaccines

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    Some neutralizing antibodies against HIV-1 envelope proteins were highly effective to inhibit the infection of different strains in vitro, and existed in the infected individuals with very low levels. We suggested multi-epitope-vaccine as a new strategy to increase levels of neutralizing antibodies and the abilities against HIV mutation in vivo. Two candidate multi-epitope-vaccines induced antibodies with predefined multi-epitope-specificity in rhesus macaque. These antibodies recognized corresponding neutralizing epitopes on epitope-peptides, gp41 peptides, V3 loop peptide, rsgp41 and rgp120. Besides, three candidate epitope-vaccines in combination (another kind of multi-epitopevaccines) showed similar potency to induce predefined multiple immune responses in rabbits. These results suggest that multi-epitope-vaccines may be a new strategy to induce multi-antiviral activities against HIV-1 infection and mutafions.

  18. Predicting population coverage of T-cell epitope-based diagnostics and vaccines

    Directory of Open Access Journals (Sweden)

    Newman Mark J

    2006-03-01

    Full Text Available Abstract Background T cells recognize a complex between a specific major histocompatibility complex (MHC molecule and a particular pathogen-derived epitope. A given epitope will elicit a response only in individuals that express an MHC molecule capable of binding that particular epitope. MHC molecules are extremely polymorphic and over a thousand different human MHC (HLA alleles are known. A disproportionate amount of MHC polymorphism occurs in positions constituting the peptide-binding region, and as a result, MHC molecules exhibit a widely varying binding specificity. In the design of peptide-based vaccines and diagnostics, the issue of population coverage in relation to MHC polymorphism is further complicated by the fact that different HLA types are expressed at dramatically different frequencies in different ethnicities. Thus, without careful consideration, a vaccine or diagnostic with ethnically biased population coverage could result. Results To address this issue, an algorithm was developed to calculate, on the basis of HLA genotypic frequencies, the fraction of individuals expected to respond to a given epitope set, diagnostic or vaccine. The population coverage estimates are based on MHC binding and/or T cell restriction data, although the tool can be utilized in a more general fashion. The algorithm was implemented as a web-application available at http://epitope.liai.org:8080/tools/population. Conclusion We have developed a web-based tool to predict population coverage of T-cell epitope-based diagnostics and vaccines based on MHC binding and/or T cell restriction data. Accordingly, epitope-based vaccines or diagnostics can be designed to maximize population coverage, while minimizing complexity (that is, the number of different epitopes included in the diagnostic or vaccine, and also minimizing the variability of coverage obtained or projected in different ethnic groups.

  19. Preparation and epitope characterization of monoclonal antibodies against firefly luciferase

    Institute of Scientific and Technical Information of China (English)

    徐沁; 丁建芳; 胡红雨; 许根俊

    1999-01-01

    The 6-His tagged firefly luciferase was highly expressed in E. coli and purified to homogeneity by affinity chromatography and gel filtration. After immunizing Balb/c mice with the antigen, 6 hybridomas clones were found to secrete monoelonal antibodies (mAbs) and the mAbs were also purified separately. The competitive binding experiments show that 2 mAbs can bind heat-denatured antigen or its proteolytic fragments but not the native lueiferase, suggesting that their epitopes might be accommodated in the internal segments of the protein. On the other hand, the other 4 mAbs are capable of binding both native and denatured antigens. It infers that their epitopes locate in the segments on the protein surface. The results also suggest that the six mAbs are all sequence-specific.

  20. Identification of an epitope of SARS-coronavirus nucleocapsid protein

    Institute of Scientific and Technical Information of China (English)

    YING LIN; JIN WANG; HONG XIA WANG; HUA LIANG JIANG; JIAN HUA SHEN; YOU HUA XIE; YUAN WANG; GANG PEI; BEI FEN SHEN; JIA RUI WU; BING SUN; XU SHEN; RUI FU YANG; YI XUE LI; YONG YONG JI; YOU YU HE; MUDE SHI; WEI LU; TIE LIU SHI

    2003-01-01

    The nucleocapsid (N) protein of severe acute respiratory syndrome-coronavirus (SARS-CoV) is a majorvirion structural protein. In this study, two epitopes (N1 and N2) of the N protein of SARS-CoV werepredicted by bioinformatics analysis. After immunization with two peptides, the peptides-specific antibodieswere isolated from the immunized rabbits. The further experiments demonstrated that N1 peptide-inducedpolyclonal antibodies had a high affinity to bind to E. coli expressed N protein of SARS-CoV. Furthermore, itwas confirmed that N1 peptide-specific IgG antibodies were detectable in the sera of severe acute respiratorysyndrome (SARS) patients. The results indicated that an epitope of the N protein has been identified andN protein specific Abs were produced by peptide immunization, which will be useful for the study of SARS-CoV.

  1. Prediction of HLA-A 2.1-restricted CTL epitopes from IGFBP7 antigen of lung carcinoma

    Institute of Scientific and Technical Information of China (English)

    Zhao Weipeng; Long Haixia; Zhu Bo; Duan Yuzhong; Chen Zhengtang

    2009-01-01

    Objective: With the development of peptide-based cancer specific immunotherapy, the prediction of CTL epitopes from insulin-like growth factor-binding protein 7 (IGFBP7) is very important for some research about tumor metastasis. Because HLA-A2.1-expressing individuals cover >50% in the population of China, we aimed at identifying IGFBP7-encoded peptide presented by HLA-A2.1. Methods: In our study, a HLA-A2.1 restricted CTL epitope was identified by using the following two-step procedure: (a) computer-based epitope prediction from the amino acid sequence of IGFBP7 antigen; (b) Validation with epitope molecular modeling. Results: We obtained four epitopes with high immunogenicity scores by all of the three algorithms, i.e., BIMAS, SYFPEITHI and IMTECH. Each of the four candidates satisfied the criteria of the HLA-A2.1-restricted CTL epitopes in molecular modeling analysis. Conclusion: The combination of BIMAS, SYFPEITHI and IMTECH method can improve the prediction efficiency and accuracy. Due to this research herein, this four epitopes have potential value for further studied, also have potential application in peptide-mediated immunotherapy. These epitopes may be useful in the design of therapeutic peptide vaccine for lung carcinoma and as immunotherapeutic strategies against lung carcinoma after identified by immunology experiment.

  2. Identification and localization of minimal MHC-restricted CD8+ T cell epitopes within the Plasmodium falciparum AMA1 protein

    Directory of Open Access Journals (Sweden)

    Sedegah Martha

    2010-08-01

    Full Text Available Abstract Background Plasmodium falciparum apical membrane antigen-1 (AMA1 is a leading malaria vaccine candidate antigen that is expressed by sporozoite, liver and blood stage parasites. Since CD8+ T cell responses have been implicated in protection against pre-erythrocytic stage malaria, this study was designed to identify MHC class I-restricted epitopes within AMA1. Methods A recombinant adenovirus serotype 5 vector expressing P. falciparum AMA1 was highly immunogenic when administered to healthy, malaria-naive adult volunteers as determined by IFN-γ ELISpot responses to peptide pools containing overlapping 15-mer peptides spanning full-length AMA1. Computerized algorithms (NetMHC software were used to predict minimal MHC-restricted 8-10-mer epitope sequences within AMA1 15-mer peptides active in ELISpot. A subset of epitopes was synthesized and tested for induction of CD8+ T cell IFN-γ responses by ELISpot depletion and ICS assays. A 3-dimensional model combining Domains I + II of P. falciparum AMA1 and Domain III of P. vivax AMA1 was used to map these epitopes. Results Fourteen 8-10-mer epitopes were predicted to bind to HLA supertypes A01 (3 epitopes, A02 (4 epitopes, B08 (2 epitopes and B44 (5 epitopes. Nine of the 14 predicted epitopes were recognized in ELISpot or ELISpot and ICS assays by one or more volunteers. Depletion of T cell subsets confirmed that these epitopes were CD8+ T cell-dependent. A mixture of the 14 minimal epitopes was capable of recalling CD8+ T cell IFN-γ responses from PBMC of immunized volunteers. Thirteen of the 14 predicted epitopes were polymorphic and the majority localized to the more conserved front surface of the AMA1 model structure. Conclusions This study predicted 14 and confirmed nine MHC class I-restricted CD8+ T cell epitopes on AMA1 recognized in the context of seven HLA alleles. These HLA alleles belong to four HLA supertypes that have a phenotypic frequency between 23% - 100% in different human

  3. 重组多价人精子表位肽的原核表达及其条件优化%Prokaryotic expression and expression condition optimizing on recombinant multi-epitopes peptide of human sperm antigens

    Institute of Scientific and Technical Information of China (English)

    谢琦; 林军; 白玲; 石青峰; 叶元; 杨冰; 刘永明

    2012-01-01

    目的 构建重组多价人精子表位肽原核表达质粒,优化其在大肠杆菌中的表达条件.方法 用化学合成法合成多价人精子表位肽基因.该基因经PCR扩增,BamHI和XhoI双酶切后,克隆至GST融合表达载体PGEX-4T-1获得重组质粒.将该重组质粒转化E.coli BL21 (DE3),经IPTG诱导表达.利用SDS-PAGE电泳和AlphaEase凝胶电泳图像分析系统,观察在摇瓶发酵条件下改变培养基组成、诱导温度、诱导时机、诱导剂浓度和诱导时间等条件对GST-多价人精子表位肽融合蛋白表达量的影响.结果 构建了重组多价人精子表位肽原核表达载体.在摇瓶实验中,优化的表达条件为:TB培养基,诱导温度37℃,在菌体对数生长的中后期进行诱导,IPTG诱导终浓度0.05 mmol/L,诱导时间5h.优化后GST-多价人精子表位肽融合蛋白的表达量占菌体总蛋白的30.2%,且主要以可溶形式表达.结论 成功构建重组多价人精子表位肽原核表达质粒,重组表达载体在E.coli BL21 (DE3)内主要表达可溶形式的GST-多价人精子表位肽融合蛋白,在优化条件下可获得较高的表达量.%OBJECTIVE To construct the prokaryotic expression recombinant plasmid PGEX-4T-l/AG10-IR9-YLP12 and optimize the expression conditions of GST- AG10-IR9-YLP12 fusion protein in E.coli. METHODS The AG10-IR9-YLP12 gene was synthesized chemically. The target gene was amplified by polymerase chain reaction ( PCR). After being digested with BamHI and Xhol, the target gene was cloned into GST fusion expression vector PGEX-4T-1. E. coli BL21 (DE3) was trans-fected with the recombinant plasmid and GST- AG10-1R9-YLP12 fusion protein was induced by IPTG. Under the shake flask fermentation conditions, the factors that may be involved in the recombinant gene expression such as culture medium, induction temperature, induction period, inducer concentration and induction time on the expression level of target protein were investigated with SDS

  4. Variable epitope library carrying heavily mutated survivin-derived CTL epitope variants as a new class of efficient vaccine immunogen tested in a mouse model of breast cancer.

    Science.gov (United States)

    NoeDominguez-Romero, Allan; Zamora-Alvarado, Rubén; Servín-Blanco, Rodolfo; Pérez-Hernández, Erendira G; Castrillon-Rivera, Laura E; Munguia, Maria Elena; Acero, Gonzalo; Govezensky, Tzipe; Gevorkian, Goar; Manoutcharian, Karen

    2014-01-01

    The antigenic variability of tumor cells leading to dynamic changes in cancer epitope landscape along with escape from immune surveillance by down-regulating tumor antigen expression/presentation and immune tolerance are major obstacles for the design of effective vaccines. We have developed a novel concept for immunogen construction based on introduction of massive mutations within the epitopes targeting antigenically variable pathogens and diseases. Previously, we showed that these immunogens carrying large combinatorial libraries of mutated epitope variants, termed as variable epitope libraries (VELs), induce potent, broad and long lasting CD8+IFN-γ+ T-cell response as well as HIV-neutralizing antibodies. In this proof-of-concept study, we tested immunogenic properties and anti-tumor effects of the VELs bearing survivin-derived CTL epitope (GWEPDDNPI) variants in an aggressive metastatic mouse 4T1 breast tumor model. The constructed VELs had complexities of 10,500 and 8,000 individual members, generated as combinatorial M13 phage display and synthetic peptide libraries, respectively, with structural composition GWXPXDXPI, where X is any of 20 natural amino acids. Statistically significant tumor growth inhibition was observed in BALB/c mice immunized with the VELs in both prophylactic and therapeutic settings. Vaccinated mice developed epitope-specific spleen cell and CD8+ IFN-γ+ T-cell responses that recognize more than 50% of the panel of 87 mutated epitope variants, as demonstrated in T-cell proliferation assays and FACS analysis. These data indicate the feasibility of the application of this new class of immunogens based on VEL concept as an alternative approach for the development of molecular vaccines against cancer.

  5. Construction and characterization of 3A-epitope-tagged foot-and-mouth disease virus.

    Science.gov (United States)

    Ma, Xueqing; Li, Pinghua; Sun, Pu; Bai, Xingwen; Bao, Huifang; Lu, Zengjun; Fu, Yuanfang; Cao, Yimei; Li, Dong; Chen, Yingli; Qiao, Zilin; Liu, Zaixin

    2015-04-01

    Nonstructural protein 3A of foot-and-mouth disease virus (FMDV) is a partially conserved protein of 153 amino acids (aa) in most FMDVs examined to date. Specific deletion in the FMDV 3A protein has been associated with the inability of FMDV to grow in primary bovine cells and cause disease in cattle. However, the aa residues playing key roles in these processes are poorly understood. In this study, we constructed epitope-tagged FMDVs containing an 8 aa FLAG epitope, a 9 aa haemagglutinin (HA) epitope, and a 10 aa c-Myc epitope to substitute residues 94-101, 93-101, and 93-102 of 3A protein, respectively, using a recently developed O/SEA/Mya-98 FMDV infectious cDNA clone. Immunofluorescence assay (IFA), Western blot and sequence analysis showed that the epitope-tagged viruses stably maintained and expressed the foreign epitopes even after 10 serial passages in BHK-21 cells. The epitope-tagged viruses displayed growth properties and plaque phenotypes similar to those of the parental virus in BHK-21 cells. However, the epitope-tagged viruses exhibited lower growth rates and smaller plaque size phenotypes than those of the parental virus in primary fetal bovine kidney (FBK) cells, but similar growth properties and plaque phenotypes to those of the recombinant viruses harboring 93-102 deletion in 3A. These results demonstrate that the decreased ability of FMDV to replicate in primary bovine cells was not associated with the length of 3A, and the genetic determinant thought to play key role in decreased ability to replicate in primary bovine cells could be reduced from 93-102 residues to 8 aa residues at positions 94-101 in 3A protein.

  6. Epitope discovery with phylogenetic hidden Markov models.

    LENUS (Irish Health Repository)

    Lacerda, Miguel

    2010-05-01

    Existing methods for the prediction of immunologically active T-cell epitopes are based on the amino acid sequence or structure of pathogen proteins. Additional information regarding the locations of epitopes may be acquired by considering the evolution of viruses in hosts with different immune backgrounds. In particular, immune-dependent evolutionary patterns at sites within or near T-cell epitopes can be used to enhance epitope identification. We have developed a mutation-selection model of T-cell epitope evolution that allows the human leukocyte antigen (HLA) genotype of the host to influence the evolutionary process. This is one of the first examples of the incorporation of environmental parameters into a phylogenetic model and has many other potential applications where the selection pressures exerted on an organism can be related directly to environmental factors. We combine this novel evolutionary model with a hidden Markov model to identify contiguous amino acid positions that appear to evolve under immune pressure in the presence of specific host immune alleles and that therefore represent potential epitopes. This phylogenetic hidden Markov model provides a rigorous probabilistic framework that can be combined with sequence or structural information to improve epitope prediction. As a demonstration, we apply the model to a data set of HIV-1 protein-coding sequences and host HLA genotypes.

  7. Automatic Generation of Validated Specific Epitope Sets

    Directory of Open Access Journals (Sweden)

    Sebastian Carrasco Pro

    2015-01-01

    Full Text Available Accurate measurement of B and T cell responses is a valuable tool to study autoimmunity, allergies, immunity to pathogens, and host-pathogen interactions and assist in the design and evaluation of T cell vaccines and immunotherapies. In this context, it is desirable to elucidate a method to select validated reference sets of epitopes to allow detection of T and B cells. However, the ever-growing information contained in the Immune Epitope Database (IEDB and the differences in quality and subjects studied between epitope assays make this task complicated. In this study, we develop a novel method to automatically select reference epitope sets according to a categorization system employed by the IEDB. From the sets generated, three epitope sets (EBV, mycobacteria and dengue were experimentally validated by detection of T cell reactivity ex vivo from human donors. Furthermore, a web application that will potentially be implemented in the IEDB was created to allow users the capacity to generate customized epitope sets.

  8. A series of N-terminal epitope tagged Hdh knock-in alleles expressing normal and mutant huntingtin: their application to understanding the effect of increasing the length of normal huntingtin’s polyglutamine stretch on CAG140 mouse model pathogenesis

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    Zheng Shuqiu

    2012-08-01

    Full Text Available Abstract Background Huntington’s disease (HD is an autosomal dominant neurodegenerative disease that is caused by the expansion of a polyglutamine (polyQ stretch within Huntingtin (htt, the protein product of the HD gene. Although studies in vitro have suggested that the mutant htt can act in a potentially dominant negative fashion by sequestering wild-type htt into insoluble protein aggregates, the role of the length of the normal htt polyQ stretch, and the adjacent proline-rich region (PRR in modulating HD mouse model pathogenesis is currently unknown. Results We describe the generation and characterization of a series of knock-in HD mouse models that express versions of the mouse HD gene (Hdh encoding N-terminal hemaglutinin (HA or 3xFlag epitope tagged full-length htt with different polyQ lengths (HA7Q-, 3xFlag7Q-, 3xFlag20Q-, and 3xFlag140Q-htt and substitution of the adjacent mouse PRR with the human PRR (3xFlag20Q- and 3xFlag140Q-htt. Using co-immunoprecipitation and immunohistochemistry analyses, we detect no significant interaction between soluble full-length normal 7Q- htt and mutant (140Q htt, but we do observe N-terminal fragments of epitope-tagged normal htt in mutant htt aggregates. When the sequences encoding normal mouse htt’s polyQ stretch and PRR are replaced with non-pathogenic human sequence in mice also expressing 140Q-htt, aggregation foci within the striatum, and the mean size of htt inclusions are increased, along with an increase in striatal lipofuscin and gliosis. Conclusion In mice, soluble full-length normal and mutant htt are predominantly monomeric. In heterozygous knock-in HD mouse models, substituting the normal mouse polyQ and PRR with normal human sequence can exacerbate some neuropathological phenotypes.

  9. Prokaryotic Expression of Major Antigenic Epitope Region of African Swine Fever Virus VP73 Protein and Preparation of Polyclonal Antibody%非洲猪瘟病毒VP73蛋白主要抗原表位区的原核表达及多克隆抗体的制备

    Institute of Scientific and Technical Information of China (English)

    李洪利; 王志亮; 王君玮; 张维; 吴晓东; 赵永刚; 李慧; 李娟; 包静月; 曹金山

    2012-01-01

    构建重组原核表达载体pET32a-VP73,将pET32a-VP73转化BL21感受态细胞,经IPTG诱导,VP73蛋白主要抗原表位区可稳定高效的表达,SDS-PAGE结果表明,IPTG终浓度为1.0 mmol/L,诱导5h蛋白质表达量最高,表达蛋白为融合蛋白,分子质量约为42 ku.蛋白质纯化后,经SDS-PAGE及Western blotting鉴定,确定表达产物为非洲猪瘟病毒VP73主要抗原表位区融合蛋白.将纯化的蛋白质免疫新西兰大白兔,免疫前后收集血清.用间接ELISA方法测定血清抗体效价,并以非洲猪瘟阳性血清、兔免疫前后血清及猪瘟、猪繁殖与呼吸综合征、猪伪狂犬病阳性血清为一抗,确定该蛋白质的特异性.结果表明,制备的抗血清效价达到1∶1024000,能与纯化的VP73主要抗原表位区蛋白质发生反应.制备的多克隆抗体为VP73蛋白的免疫学研究和非洲猪瘟血清学诊断奠定了基础.%Constructed recombinant prokaryotic expression vector pET32a-VP73 was transformed into E, colt BL21, major antigenic epitope region of VP73 protein was induced stably and efficiently by IPTG, the result of SDS-PAGE showed, when final concentration of IPTG was 1. 0 mmol/L, and pET32a-VP73 was induced 5 h, expression of major antigenic epitope region of VP73 protein was highest, the expressed protein was fusion protein, molecular weight was approximately 42 ku. The protein was identified as major antigenic epitope region of VP73 protein by SDS-PAGE and Western blotting. The purified major antigenic epitope region of VP73 protein was injected to New Zealand rabbits, serum was collected before and after injection. Antibody titer was determined by ELISA. Specificity of this protein was determined by ELISA whose primary antibody were ASF positive serum, rabbits serum before and after injection and positive serum of CSF, PRRS, pig pseudorabies. The result showed the titer of antiserum reached up to 1 : 1024000, and gave rise to reactions with VP73 major antigenic region

  10. Discovery of common marburgvirus protective epitopes in a BALB/c mouse model

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    Olinger Gene G

    2009-08-01

    Full Text Available Abstract Background Marburg virus (MARV causes acute hemorrhagic fever that is often lethal, and no licensed vaccines are available for preventing this deadly viral infection. The immune mechanisms for protection against MARV are poorly understood, but previous studies suggest that both antibodies and T cells are required. In our study, we infected BALB/c mice with plaque-purified, nonlethal MARV and used overlapping peptides to map H2d-restricted CD8+ T-cell epitopes. Methods Splenocytes from mice infected with nonlethal MARV were harvested and stimulated with multiple overlapping 15-mer peptide pools, and reactive CD8+ T cells were evaluated for antigen specificity by measuring upregulation of CD44 and interferon-γ expression. After confirming positive reactivity to specific 15-mer peptides, we used extrapolated 9-mer epitopes to evaluate the induction of cytotoxic T-cell responses and protection from lethal MARV challenge in BALB/c mice. Results We discovered a CD8+ T-cell epitope within both the MARV glycoprotein (GP and nucleoprotein (NP that triggered cytotoxic T-cell responses. These responses were also protective when epitope-specific splenocytes were transferred into naïve animals. Conclusion Epitope mapping of MARV GP, NP, and VP40 provides the first evidence that specific MARV-epitope induction of cellular immune responses is sufficient to combat infection. Establishment of CD8+ T-cell epitopes that are reactive to MARV proteins provides an important research tool for dissecting the significance of cellular immune responses in BALB/c mice infected with MARV.

  11. Rapid identification of novel immunodominant proteins and characterization of a specific linear epitope of Campylobacter jejuni.

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    Sebastian Hoppe

    Full Text Available Campylobacter jejuni remains one of the major gut pathogens of our time. Its zoonotic nature and wide-spread distribution in industrialized countries calls for a quick and reliable diagnostic tool. Antibody-based detection presents a suitable means to identify pathogenic bacteria. However, the knowledge about immunodominant targets is limited. Thus, an approach is presented, which allows for the rapid screening of numerous cDNA derived expression clones to identify novel antigens. The deeper understanding of immunodominant proteins assists in the design of diagnostic tools and furthers the insight into the bacterium's pathogenicity as well as revealing potential candidates for vaccination. We have successfully screened 1536 clones of an expression library to identify 22 proteins that have not been described as immunodominant before. After subcloning the corresponding 22 genes and expression of full-length proteins, we investigated the immunodominant character by microarrays and ELISA. Subsequently, seven proteins were selected for epitope mapping. For cj0669 and cj0920c linear epitopes were identified. For cj0669, specificity assays revealed a specific linear epitope site. Consequently, an eleven amino acid residue sequence TLIKELKRLGI was analyzed via alanine scan, which revealed the glycine residue to be significant for binding of the antibody. The innovative approach presented herein of generating cDNAs of prokaryotes in combination with a microarray platform rendering time-consuming purification steps obsolete has helped to illuminate novel immunodominant proteins of C.jejuni. The findings of a specific linear epitope pave the way for a plethora of future research and the potential use in diagnostic applications such as serological screenings. Moreover, the current approach is easily adaptable to other highly relevant bacteria making it a formidable tool for the future discovery of antigens and potential biomarkers. Consequently, it is

  12. Rapid Identification of Novel Immunodominant Proteins and Characterization of a Specific Linear Epitope of Campylobacter jejuni

    Science.gov (United States)

    Hoppe, Sebastian; Bier, Frank F.; Nickisch-Rosenegk, Markus v.

    2013-01-01

    Campylobacter jejuni remains one of the major gut pathogens of our time. Its zoonotic nature and wide-spread distribution in industrialized countries calls for a quick and reliable diagnostic tool. Antibody-based detection presents a suitable means to identify pathogenic bacteria. However, the knowledge about immunodominant targets is limited. Thus, an approach is presented, which allows for the rapid screening of numerous cDNA derived expression clones to identify novel antigens. The deeper understanding of immunodominant proteins assists in the design of diagnostic tools and furthers the insight into the bacterium’s pathogenicity as well as revealing potential candidates for vaccination. We have successfully screened 1536 clones of an expression library to identify 22 proteins that have not been described as immunodominant before. After subcloning the corresponding 22 genes and expression of full-length proteins, we investigated the immunodominant character by microarrays and ELISA. Subsequently, seven proteins were selected for epitope mapping. For cj0669 and cj0920c linear epitopes were identified. For cj0669, specificity assays revealed a specific linear epitope site. Consequently, an eleven amino acid residue sequence TLIKELKRLGI was analyzed via alanine scan, which revealed the glycine residue to be significant for binding of the antibody. The innovative approach presented herein of generating cDNAs of prokaryotes in combination with a microarray platform rendering time-consuming purification steps obsolete has helped to illuminate novel immunodominant proteins of C.jejuni. The findings of a specific linear epitope pave the way for a plethora of future research and the potential use in diagnostic applications such as serological screenings. Moreover, the current approach is easily adaptable to other highly relevant bacteria making it a formidable tool for the future discovery of antigens and potential biomarkers. Consequently, it is desirable to simplify

  13. Intramolecular epitope spreading in Heymann nephritis.

    Science.gov (United States)

    Shah, Pallavi; Tramontano, Alfonso; Makker, Sudesh P

    2007-12-01

    Immunization with megalin induces active Heymann nephritis, which reproduces features of human idiopathic membranous glomerulonephritis. Megalin is a complex immunological target with four discrete ligand-binding domains (LBDs) that may contain epitopes to which pathogenic autoantibodies are directed. Recently, a 236-residue N-terminal fragment, termed "L6," that spans the first LBD was shown to induce autoantibodies and severe disease. We used this model to examine epitope-specific contributions to pathogenesis. Sera obtained from rats 4 weeks after immunization with L6 demonstrated reactivity only with the L6 fragment on Western blot, whereas sera obtained after 8 weeks demonstrated reactivity with all four recombinant fragments of interest (L6 and LBDs II, III, and IV). We demonstrated that the L6 immunogen does not contain the epitopes responsible for the reactivity to the LBD fragments. Therefore, the appearance of antibodies directed at LBD fragments several weeks after the primary immune response suggests intramolecular epitope spreading. In vivo, we observed a temporal association between increased proteinuria and the appearance of antibodies to LBD fragments. These data implicate B cell epitope spreading in antibody-mediated pathogenesis of active Heymann nephritis, a model that should prove valuable for further study of autoimmune dysregulation.

  14. Oxidation-specific epitopes are dominant targets of innate natural antibodies in mice and humans.

    Science.gov (United States)

    Chou, Meng-Yun; Fogelstrand, Linda; Hartvigsen, Karsten; Hansen, Lotte F; Woelkers, Douglas; Shaw, Peter X; Choi, Jeomil; Perkmann, Thomas; Bäckhed, Fredrik; Miller, Yury I; Hörkkö, Sohvi; Corr, Maripat; Witztum, Joseph L; Binder, Christoph J

    2009-05-01

    Atherosclerosis is a chronic inflammatory disease characterized by the accumulation of oxidized lipoproteins and apoptotic cells. Adaptive immune responses to various oxidation-specific epitopes play an important role in atherogenesis. However, accumulating evidence suggests that these epitopes are also recognized by innate receptors, such as scavenger receptors on macrophages, and plasma proteins, such as C-reactive protein (CRP). Here, we provide multiple lines of evidence that oxidation-specific epitopes constitute a dominant, previously unrecognized target of natural Abs (NAbs) in both mice and humans. Using reconstituted mice expressing solely IgM NAbs, we have shown that approximately 30% of all NAbs bound to model oxidation-specific epitopes, as well as to atherosclerotic lesions and apoptotic cells. Because oxidative processes are ubiquitous, we hypothesized that these epitopes exert selective pressure to expand NAbs, which in turn play an important role in mediating homeostatic functions consequent to inflammation and cell death, as demonstrated by their ability to facilitate apoptotic cell clearance. These findings provide novel insights into the functions of NAbs in mediating host homeostasis and into their roles in health and diseases, such as chronic inflammatory diseases and atherosclerosis.

  15. Equivalent T cell epitope promiscuity in ecologically diverse human pathogens.

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    Kirsten E Wiens

    Full Text Available BACKGROUND: The HLA (human leukocyte antigen molecules that present pathogen-derived epitopes to T cells are highly diverse. Correspondingly, many pathogens such as HIV evolve epitope variants in order to evade immune recognition. In contrast, another persistent human pathogen, Mycobacterium tuberculosis, has highly conserved epitope sequences. This raises the question whether there is also a difference in the ability of these pathogens' epitopes to bind diverse HLA alleles, referred to as an epitope's binding promiscuity. To address this question, we compared the in silico HLA binding promiscuity of T cell epitopes from pathogens with distinct infection strategies and outcomes of human exposure. METHODS: We used computer algorithms to predict the binding affinity of experimentally-verified microbial epitope peptides to diverse HLA-DR, HLA-A and HLA-B alleles. We then analyzed binding promiscuity of epitopes derived from HIV and M. tuberculosis. We also analyzed promiscuity of epitopes from Streptococcus pyogenes, which is known to exhibit epitope diversity, and epitopes of Bacillus anthracis and Clostridium tetani toxins, as these bacteria do not depend on human hosts for their survival or replication, and their toxin antigens are highly immunogenic human vaccines. RESULTS: We found that B. anthracis and C. tetani epitopes were the most promiscuous of the group that we analyzed. However, there was no consistent difference or trend in promiscuity in epitopes contained in HIV, M. tuberculosis, and S. pyogenes. CONCLUSIONS: Our results show that human pathogens with distinct immune evasion strategies and epitope diversities exhibit equivalent levels of T cell epitope promiscuity. These results indicate that differences in epitope promiscuity do not account for the observed differences in epitope variation and conservation.

  16. In silico cloning and B/T cell epitope prediction of triosephosphate isomerase from Echinococcus granulosus.

    Science.gov (United States)

    Wang, Fen; Ye, Bin

    2016-10-01

    Cystic echinococcosis is a worldwide zoonosis caused by Echinococcus granulosus. Because the methods of diagnosis and treatment for cystic echinococcosis were limited, it is still necessary to screen target proteins for the development of new anti-hydatidosis vaccine. In this study, the triosephosphate isomerase gene of E. granulosus was in silico cloned. The B cell and T cell epitopes were predicted by bioinformatics methods. The cDNA sequence of EgTIM was composition of 1094 base pairs, with an open reading frame of 753 base pairs. The deduced amino acid sequences were composed of 250 amino acids. Five cross-reactive epitopes, locating on 21aa-35aa, 43aa-57aa, 94aa-107aa, 115-129aa, and 164aa-183aa, could be expected to serve as candidate epitopes in the development of vaccine against E. granulosus. These results could provide bases for gene cloning, recombinant expression, and the designation of anti-hydatidosis vaccine.

  17. Virus-Like Particles of Chimeric Recombinant Porcine Circovirus Type 2 as Antigen Vehicle Carrying Foreign Epitopes

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    Huawei Zhang

    2014-12-01

    Full Text Available Virus-like particles (VLPs of chimeric porcine circovirus type 2 (PCV2 were generated by replacing the nuclear localization signal (NLS; at 1–39 aa of PCV2 capsid protein (Cap with classical swine fever virus (CSFV T-cell epitope (1446–1460 aa, CSFV B-cell epitope (693–716 aa and CSFV T-cell epitope conjugated with B-cell epitope. The recombinant proteins were expressed using the baculovirus expression system and detected by immunoblotting and indirect immunofluorescence assay. The abilities to form PCV2 VLPs were confirmed by transmission electron microscopy. Immunogenicities of the three recombinant proteins were evaluated in mice. Our Results indicated that Cap protein NLS deletion or substitution with CSFV epitopes did not affect the VLPs assembly. Three chimeric Cap proteins could form VLPs and induce efficient humoral and cellular immunity against PCV2 and CSFV in mice. Results show that PCV2 VLPs can be used as an efficient antigen carrier for delivery of foreign epitopes, and a potential novel vaccine.

  18. Identification of a conserved B-cell epitope on reticuloendotheliosis virus envelope protein by screening a phage-displayed random peptide library.

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    Mei Xue

    Full Text Available BACKGROUND: The gp90 protein of avian reticuloendotheliosis-associated virus (REV-A is an important envelope glycoprotein, which is responsible for inducing protective antibody immune responses in animals. B-cell epitopes on the gp90 protein of REV have not been well studied and reported. METHODS AND RESULTS: This study describes the identification of a linear B-cell epitope on the gp90 protein by screening a phage-displayed 12-mer random peptide library with the neutralizing monoclonal antibody (mAb A9E8 directed against the gp90. The mAb A9E8 recognized phages displaying peptides with the consensus motif SVQYHPL. Amino acid sequence of the motif exactly matched (213SVQYHPL(219 of the gp90. Further identification of the displayed B cell epitope was conducted using a set of truncated peptides expressed as GST fusion proteins and the Western blot results indicated that (213SVQYHPL(219 was the minimal determinant of the linear B cell epitope recognized by the mAb A9E8. Moreover, an eight amino acid peptide SVQYHPLA was proven to be the minimal unit of the epitope with the maximal binding activity to mAb A9E8. The REV-A-positive chicken serum reacted with the minimal linear epitopes in Western blot, revealing the importance of the eight amino acids of the epitope in antibody-epitope binding activity. Furthermore, we found that the epitope is a common motif shared among REV-A and other members of REV group. CONCLUSIONS AND SIGNIFICANCE: We identified (213SVQYHPL(219 as a gp90-specific linear B-cell epitope recognized by the neutralizing mAb A9E8. The results in this study may have potential applications in development of diagnostic techniques and epitope-based marker vaccines against REV-A and other viruses of the REV group.

  19. Immune epitope database analysis resource (IEDB-AR)

    DEFF Research Database (Denmark)

    Zhang, Qing; Wang, Peng; Kim, Yohan;

    2008-01-01

    We present a new release of the immune epitope database analysis resource (IEDB-AR, http://tools.immuneepitope.org), a repository of web-based tools for the prediction and analysis of immune epitopes. New functionalities have been added to most of the previously implemented tools, and a total...... of eight new tools were added, including two B-cell epitope prediction tools, four T-cell epitope prediction tools and two analysis tools....

  20. Characterization of T cell epitopes in bovine α-lactalbumin

    NARCIS (Netherlands)

    Meulenbroek, Laura A P M; den Hartog Jager, Constance F; Lebens, Ans F M; Knulst, André C; Bruijnzeel-Koomen, Carla A F M; Garssen, Johan; Knippels, Léon M J; van Hoffen, Els

    2014-01-01

    BACKGROUND: Recent studies have indicated that peptides containing T cell epitopes may be used for immunotherapy. While for several cow's milk allergens the T cell epitopes have been described, the T cell epitopes in the major allergen α-lactalbumin (α-LAC) are unknown. Therefore, the aim of this st

  1. Design, Expression and Identification of Specific Multi-Epitope Antigen of Campylobacter Jejuni Surface Proteins%空肠弯曲菌表面蛋白特异性多表位抗原的设计、表达与鉴定

    Institute of Scientific and Technical Information of China (English)

    欧瑜; 鄢方兵; 唐泰山; 祝长青; 蒋原; 张常印

    2012-01-01

    构建空肠弯曲菌表面蛋白特异性多表位抗原原核表达载体,并在大肠杆菌中诱导表达,免疫动物后检测其抗血清与空肠弯曲菌菌体的反应性.从空肠弯曲菌的6个表面蛋白中分析到特异性抗原表位,人工合成其串联基因片段,将其插入原核表达载体,获得重组质粒pET-32a(+ )-CJMEA-A,经IPTG诱导后表达出25k的目标蛋白,纯化后免疫新西兰大白兔,Western blot和间接ELISA检测重组蛋白抗原性和抗血清反应性.结果表明目标蛋白具有良好的反应原和免疫原性,抗血清能与菌体发生反应.其抗体可用于C.jejuni免疫磁珠和免疫胶体金等检测方法的建立.%To construct and to express the gene of specific multi-epitope antigen from Campylobacter jejuni and to detect the reactivity of its antibody to this bacteria. Firstly ,some specific antigenic determinants were obtained after 6 surface proteins of Campytobacter jejuni were analyzed. Secondly these epitopes were linked in series and its gene was synthesized. Thirdly, the recombinant plasmid pET-32a( + )-CJMEA-A was obtained when the gene was inserted into pET-32a( + ) vector. Then,after being induced by IPTG,the 25ku of fusion protein was expressed,purified and used for the immunization of rabbits. Finally,SDS-PAGE and indirect ELISA were used to detect the antigenicity of the protein and the reactivity of the antiserum. The results showed that this protein was expressed successfully;it possesses good unnuinogem'city and reactogenicity;its antiserum can react to thalli of C. Jejuni . A conclusion can be made that the antibody of this protein could be used to detect C. Jejuni by inununomagnetic beads or immune colloidal gold technique.

  2. Development of an epitope conservancy analysis tool to facilitate the design of epitope-based diagnostics and vaccines

    Directory of Open Access Journals (Sweden)

    Fusseder Nicolas

    2007-09-01

    Full Text Available Abstract Background In an epitope-based vaccine setting, the use of conserved epitopes would be expected to provide broader protection across multiple strains, or even species, than epitopes derived from highly variable genome regions. Conversely, in a diagnostic and disease monitoring setting, epitopes that are specific to a given pathogen strain, for example, can be used to monitor responses to that particular infectious strain. In both cases, concrete information pertaining to the degree of conservancy of the epitope(s considered is crucial. Results To assist in the selection of epitopes with the desired degree of conservation, we have developed a new tool to determine the variability of epitopes within a given set of protein sequences. The tool was implemented as a component of the Immune Epitope Database and Analysis Resources (IEDB, and is directly accessible at http://tools.immuneepitope.org/tools/conservancy. Conclusion An epitope conservancy analysis tool was developed to analyze the variability or conservation of epitopes. The tool is user friendly, and is expected to aid in the design of epitope-based vaccines and diagnostics.

  3. Neutralization epitopes on HIV pseudotyped with HTLV-I: Conservation of carbohydrate Epitopes

    DEFF Research Database (Denmark)

    Sørensen, A M; Nielsen, C; Arendrup, M

    1994-01-01

    for pseudotypes to escape neutralization by the immune system in vivo. Previous reports have suggested that carbohydrate structures may be conserved neutralization epitopes on retroviruses. In this study, the neutralizing capacity of lectins and anti-carbohydrate monoclonal antibodies was found to block infection...... by cell-free pseudotypes in CD4-negative cells. We suggest that although viral cofactors might expand the tropism of HIV in vivo, HIV and HTLV-I seem to induce common carbohydrate neutralization epitopes....

  4. IgE-binding epitopes: a reappraisal

    NARCIS (Netherlands)

    R.C. Aalberse; R. Crameri

    2011-01-01

    Here, we discuss various questions related to IgE epitopes: What are the technical possibilities and pitfalls, what is currently known, how can we put this information into hypothetical frameworks and the unavoidable question: how useful is this information for patient care or allergenicity predicti

  5. Viral O-GalNAc peptide epitopes

    DEFF Research Database (Denmark)

    Olofsson, Sigvard; Blixt, Klas Ola; Bergström, Tomas

    2016-01-01

    on a novel three-step procedure that identifies any reactive viral O-glycosyl peptide epitope with respect to (i) relevant peptide sequence, (ii) the reactive glycoform out of several possible glycopeptide isomers of that peptide sequence, and (iii) possibly tolerated carbohydrate or peptide structural...

  6. Induction of epitope-specific neutralizing antibodies against West Nile virus.

    Science.gov (United States)

    Oliphant, Theodore; Nybakken, Grant E; Austin, S Kyle; Xu, Qing; Bramson, Jonathan; Loeb, Mark; Throsby, Mark; Fremont, Daved H; Pierson, Theodore C; Diamond, Michael S

    2007-11-01

    Previous studies have established that an epitope on the lateral ridge of domain III (DIII-lr) of West Nile virus (WNV) envelope (E) protein is recognized by strongly neutralizing type-specific antibodies. In contrast, an epitope against the fusion loop in domain II (DII-fl) is recognized by flavivirus cross-reactive antibodies with less neutralizing potential. Using gain- and loss-of-function E proteins and wild-type and variant WNV reporter virus particles, we evaluated the expression pattern and activity of antibodies against the DIII-lr and DII-fl epitopes in mouse and human serum after WNV infection. In mice, immunoglobulin M (IgM) antibodies to the DIII-lr epitope were detected at low levels at day 6 after infection. However, compared to IgG responses against other epitopes in DI and DII, which were readily detected at day 8, the development of IgG against DIII-lr epitope was delayed and did not appear consistently until day 15. This late time point is notable since almost all death after WNV infection in mice occurs by day 12. Nonetheless, at later time points, DIII-lr antibodies accumulated and comprised a significant fraction of the DIII-specific IgG response. In sera from infected humans, DIII-lr antibodies were detected at low levels and did not correlate with clinical outcome. In contrast, antibodies to the DII-fl were detected in all human serum samples and encompassed a significant percentage of the anti-E protein response. Our experiments suggest that the highly neutralizing DIII-lr IgG antibodies have little significant role in primary infection and that the antibody response of humans may be skewed toward the induction of cross-reactive, less-neutralizing antibodies.

  7. Induction of Epitope-Specific Neutralizing Antibodies against West Nile Virus▿

    Science.gov (United States)

    Oliphant, Theodore; Nybakken, Grant E.; Austin, S. Kyle; Xu, Qing; Bramson, Jonathan; Loeb, Mark; Throsby, Mark; Fremont, Daved H.; Pierson, Theodore C.; Diamond, Michael S.

    2007-01-01

    Previous studies have established that an epitope on the lateral ridge of domain III (DIII-lr) of West Nile virus (WNV) envelope (E) protein is recognized by strongly neutralizing type-specific antibodies. In contrast, an epitope against the fusion loop in domain II (DII-fl) is recognized by flavivirus cross-reactive antibodies with less neutralizing potential. Using gain- and loss-of-function E proteins and wild-type and variant WNV reporter virus particles, we evaluated the expression pattern and activity of antibodies against the DIII-lr and DII-fl epitopes in mouse and human serum after WNV infection. In mice, immunoglobulin M (IgM) antibodies to the DIII-lr epitope were detected at low levels at day 6 after infection. However, compared to IgG responses against other epitopes in DI and DII, which were readily detected at day 8, the development of IgG against DIII-lr epitope was delayed and did not appear consistently until day 15. This late time point is notable since almost all death after WNV infection in mice occurs by day 12. Nonetheless, at later time points, DIII-lr antibodies accumulated and comprised a significant fraction of the DIII-specific IgG response. In sera from infected humans, DIII-lr antibodies were detected at low levels and did not correlate with clinical outcome. In contrast, antibodies to the DII-fl were detected in all human serum samples and encompassed a significant percentage of the anti-E protein response. Our experiments suggest that the highly neutralizing DIII-lr IgG antibodies have little significant role in primary infection and that the antibody response of humans may be skewed toward the induction of cross-reactive, less-neutralizing antibodies. PMID:17715236

  8. Design based on Bioinformatics and Expression Optimization of Fusion Protein with Cholera Toxin B Subunit and an Epitope Peptide from Helicobacter pylori Urease A Subunit%幽门螺旋杆菌尿素酶A亚基抗原表位肽与霍乱毒素B 亚基融合蛋白的生物信息学设计及表达优化研究

    Institute of Scientific and Technical Information of China (English)

    郭乐; 刘昆梅; 李小康; 汤锋; 奚涛

    2013-01-01

      幽门螺旋杆菌(Hp)是慢性胃炎、消化性溃疡和胃癌的重要致病因子.研发基于Hp尿素酶的表位疫苗是一种很有前景的防治Hp感染的策略.本研究主要通过生物信息学软件对Hp尿素酶表位肽U21和霍乱毒素B 亚基(CTB)的连接顺序和间隔序列加以分析,设计出一种由U21和CTB构成的Hp表位疫苗CTB-UA.通过基因克隆技术构建含有融合基因CTB-UA的重组表达载体pETCUA及其重组菌株;重组菌株经经蛋白表达和优化后,利用Ni-NTP 镍离子亲和层析和DEAE Sepharose FF阴离子交换层析纯化融合蛋白CTB-UA,获得高纯度(94.8%)的融合蛋白CTB-UA.并进一步通过腹腔注射免疫BALB/ c小鼠,鉴定Hp 表位疫苗CTB-UA 的免疫学活性,经间接ELISA 鉴定小鼠能够产生针对CTB和表位肽U21的高滴度特异性抗体.结果证明,Hp表位疫苗CTB-UA 具有科学合理的结构,能在大肠杆菌表达系统中获得较高水平的表达,且具有较高的免疫学特异性,为研发防治Hp感染的表位疫苗奠定一定的实验基础.%Helicobacter pylori (Hp)is associated with the development of chronic gastritis,peptic ulcer and gastric cancer. Epitope vaccine based on the enzyme urease of Hp is a promising option for prophylactic and therapeutic vaccination against Hp infection. An epitope vaccine CTB-UA composed of cholera toxin B subunit (CTB)and an epitope peptide named U21 from urease A subunit was constructed by analyzing the coupling sequence and linker between CTB and U21 using bioinformatics software. The recombinant expression vector pETCUA containing the fusion gene CTB-UA and its recombinant strain were constructed by gene cloning technology. After protein expression and optimization,the recombinant protein CTB-UA was purified by Ni2 + -charged column chromatography and anion-exchange chromatography using DEAE sepharose FF,about 51 mg of pure target protein was obtained from 1 L of fermentation broth and the purity of CTB

  9. Evaluation of a multi-epitope subunit vaccine against avian leukosis virus subgroup J in chickens.

    Science.gov (United States)

    Xu, Qingqing; Ma, Xingjiang; Wang, Fangkun; Li, Hongmei; Zhao, Xiaomin

    2015-12-02

    The intricate sequence and antigenic variability of avian leukosis virus subgroup J (ALV-J) have led to unprecedented difficulties in the development of vaccines. Much experimental evidence demonstrates that ALV-J mutants have caused immune evasion and pose a challenge for traditional efforts to develop effective vaccines. To investigate the potential of a multi-epitope vaccination strategy to prevent chickens against ALV-J infections, a recombinant chimeric multi-epitope protein X (rCMEPX) containing both immunodominant B and T epitope concentrated domains selected from the major structural protein of ALV-J using bioinformatics approach was expressed in Escherichia coli Rosetta (DE3). Its immunogenicity and protective efficacy was studied in chickens. The results showed that rCMEPX could elicit neutralizing antibodies and cellular responses, and antibodies induced by rCMEPX could specifically recognize host cell naturally expressed ALV-J proteins, which indicated that the rCMEPX is a good immunogen. Challenge experiments showed 80% chickens that received rCMEPX were well protected against ALV-J challenge. This is the first report of a chimeric multi-epitope protein as a potential immunogen against ALV-J.

  10. Immunogenicity of a Multi-Epitope DNA Vaccine Encoding Epitopes from Cu–Zn Superoxide Dismutase and Open Reading Frames of Brucella abortus in Mice

    Science.gov (United States)

    Escalona, Emilia; Sáez, Darwin; Oñate, Angel

    2017-01-01

    Brucellosis is a bacterial zoonotic disease affecting several mammalian species that is transmitted to humans by direct or indirect contact with infected animals or their products. In cattle, brucellosis is almost invariably caused by Brucella abortus. Live, attenuated Brucella vaccines are commonly used to prevent illness in cattle, but can cause abortions in pregnant animals. It is, therefore, desirable to design an effective and safer vaccine against Brucella. We have used specific Brucella antigens that induce immunity and protection against B. abortus. A novel recombinant multi-epitope DNA vaccine specific for brucellosis was developed. To design the vaccine construct, we employed bioinformatics tools to predict epitopes present in Cu–Zn superoxide dismutase and in the open reading frames of the genomic island-3 (BAB1_0260, BAB1_0270, BAB1_0273, and BAB1_0278) of Brucella. We successfully designed a multi-epitope DNA plasmid vaccine chimera that encodes and expresses 21 epitopes. This DNA vaccine induced a specific humoral and cellular immune response in BALB/c mice. It induced a typical T-helper 1 response, eliciting production of immunoglobulin G2a and IFN-γ particularly associated with the Th1 cell subset of CD4+ T cells. The production of IL-4, an indicator of Th2 activation, was not detected in splenocytes. Therefore, it is reasonable to suggest that the vaccine induced a predominantly Th1 response. The vaccine induced a statistically significant level of protection in BALB/c mice when challenged with B. abortus 2308. This is the first use of an in silico strategy to a design a multi-epitope DNA vaccine against B. abortus. PMID:28232837

  11. B cell epitope spreading: mechanisms and contribution to autoimmune diseases.

    Science.gov (United States)

    Cornaby, Caleb; Gibbons, Lauren; Mayhew, Vera; Sloan, Chad S; Welling, Andrew; Poole, Brian D

    2015-01-01

    While a variety of factors act to trigger or initiate autoimmune diseases, the process of epitope spreading is an important contributor in their development. Epitope spreading is a diversification of the epitopes recognized by the immune system. This process happens to both T and B cells, with this review focusing on B cells. Such spreading can progress among multiple epitopes on a single antigen, or from one antigenic molecule to another. Systemic lupus erythematosus, multiple sclerosis, pemphigus, bullous pemphigoid and other autoimmune diseases, are all influenced by intermolecular and intramolecular B cell epitope spreading. Endocytic processing, antigen presentation, and somatic hypermutation act as molecular mechanisms that assist in driving epitope spreading and broadening the immune response in autoimmune diseases. The purpose of this review is to summarize our current understanding of B cell epitope spreading with regard to autoimmunity, how it contributes during the progression of various autoimmune diseases, and treatment options available.

  12. The GCTM-5 Epitope Associated with the Mucin-Like Glycoprotein FCGBP Marks Progenitor Cells in Tissues of Endodermal Origin

    Science.gov (United States)

    Stamp, Lincon A.; Braxton, David R.; Wu, Jun; Akopian, Veronika; Hasegawa, Kouichi; Chandrasoma, Parakrama T.; hawes, Susan M.; Mclean, Catriona; Petrovic, Lydia m.; WANG, KASPER; Pera, Martin F.

    2013-01-01

    Monoclonal antibodies against cell surface markers are powerful tools in the study of tissue regeneration, repair, and neoplasia, but there is a paucity of specific reagents to identify stem and progenitor cells in tissues of endodermal origin. The epitope defined by the GCTM-5 monoclonal antibody is a putative marker of hepatic progenitors. We sought to analyze further the distribution of the GCTM-5 antigen in normal tissues and disease states and to characterize the antigen biochemically. The GCTM-5 epitope was specifically expressed on tissues derived from the definitive endoderm, in particular the fetal gut, liver, and pancreas. Antibody reactivity was detected in subpopulations of normal adult biliary and pancreatic duct cells, and GCTM-5-positive cells isolated from the nonparenchymal fraction of adult liver expressed markers of progenitor cells. The GCTM-5-positive cell populations in liver and pancreas expanded greatly in numbers in disease states such as biliary atresia, cirrhosis, and pancreatitis. Neoplasms arising in these tissues also expressed the GCTM-5 antigen, with pancreatic adenocarcinoma in particular showing strong and consistent reactivity. The GCTM-5 epitope was also strongly displayed on cells undergoing intestinal metaplasia in Barrett’s esophagus, a precursor to esophageal carcinoma. Biochemical, mass spectrometry, and immunochemical studies revealed that the GCTM-5 epitope is associated with the mucin-like glycoprotein FCGBP. The GCTM-5 epitope on the mucin-like glycoprotein FCGBP is a cell surface marker for the study of normal differentiation lineages, regeneration, and disease progression in tissues of endodermal origin. PMID:22761039

  13. Effects of type II collagen epitope carbamylation and citrullination in human leucocyte antigen (HLA)-DR4(+) monozygotic twins discordant for rheumatoid arthritis.

    Science.gov (United States)

    De Santis, M; Ceribelli, A; Cavaciocchi, F; Generali, E; Massarotti, M; Isailovic, N; Crotti, C; Scherer, H U; Montecucco, C; Selmi, C

    2016-09-01

    The aim of this study is to investigate the effect of the native, citrullinated or carbamylated type II human collagen T cell- and B cell-epitopes on the adaptive immune response in rheumatoid arthritis (RA). Peripheral blood T and B cells obtained from a human leucocyte D4-related (antigen DR4(-) HLA-DR4)(+) woman with early RA, her healthy monozygotic twin and an unrelated HLA-DR3(+) woman with early RA were analysed for activation (CD154/CD69), apoptosis (annexin/7-aminoactinomycin), cytokine production [interferon (IFN)γ/interleukin (IL)-17/IL-4/IL-10/IL-6] and functional phenotype (CD45Ra/CCR7) after stimulation with the collagen native T cell epitope (T261-273), the K264 carbamylated T cell epitope (carT261-273), the native B cell epitope (B359-369) or the R360 citrullinated B cell epitope (citB359-369), and the combinations of these. The T cell memory compartment was activated by T cell epitopes in both discordant DR4(+) twins, but not in the DR3(+) RA. The collagen-specific activation of CD4(+) T cells was induced with both the native and carbamylated T cell epitopes only in the RA twin. Both T cell epitopes also induced IL-17 production in the RA twin, but a greater IL-4 and IL-10 response in the healthy twin. The citrullinated B cell epitope, particularly when combined with the carbamylated T cell epitope, induced B cell activation and an increased IL-6/IL-10 ratio in the RA twin compared to a greater IL-10 production in the healthy twin. Our data suggest that circulating collagen-specific T and B cells are found in HLA-DR4(+) subjects, but only RA activated cells express co-stimulatory molecules and produce proinflammatory cytokines. Carbamylation and citrullination further modulate the activation and cytokine polarization of T and B cells.

  14. Subdominant/cryptic CD8 T cell epitopes contribute to resistance against experimental infection with a human protozoan parasite.

    Directory of Open Access Journals (Sweden)

    Mariana R Dominguez

    Full Text Available During adaptive immune response, pathogen-specific CD8(+ T cells recognize preferentially a small number of epitopes, a phenomenon known as immunodominance. Its biological implications during natural or vaccine-induced immune responses are still unclear. Earlier, we have shown that during experimental infection, the human intracellular pathogen Trypanosoma cruzi restricts the repertoire of CD8(+ T cells generating strong immunodominance. We hypothesized that this phenomenon could be a mechanism used by the parasite to reduce the breath and magnitude of the immune response, favoring parasitism, and thus that artificially broadening the T cell repertoire could favor the host. Here, we confirmed our previous observation by showing that CD8(+ T cells of H-2(a infected mice recognized a single epitope of an immunodominant antigen of the trans-sialidase super-family. In sharp contrast, CD8(+ T cells from mice immunized with recombinant genetic vaccines (plasmid DNA and adenovirus expressing this same T. cruzi antigen recognized, in addition to the immunodominant epitope, two other subdominant epitopes. This unexpected observation allowed us to test the protective role of the immune response to subdominant epitopes. This was accomplished by genetic vaccination of mice with mutated genes that did not express a functional immunodominant epitope. We found that these mice developed immune responses directed solely to the subdominant/cryptic CD8 T cell epitopes and a significant degree of protective immunity against infection mediated by CD8(+ T cells. We concluded that artificially broadening the T cell repertoire contributes to host resistance against infection, a finding that has implications for the host-parasite relationship and vaccine development.

  15. Cloning of Gfrα1 Partial cDNA in Sheep and Prokaryotic Expression of Epitope Polypeptide%绵羊Gfrα1基因的部分cDNA克隆与抗原表位多肽的原核表达

    Institute of Scientific and Technical Information of China (English)

    刘燕; 罗奋华; 包佳婧; 刘林洪; 单飞彪; 吴应积

    2012-01-01

    旨在克隆绵羊Gfrα1基因序列,深入研究绵羊精原干细胞(SSCs).本研究通过RT-PCR扩增和分子克隆的方法克隆到了绵羊Gfrα1基因的编码区大部分片段.结果,克隆得到的绵羊Gfrα1基因的大部分cDNA序列长1 312 bp,包括1 311 bp的开放阅读框(ORF),编码437个氨基酸,与牛、人、黑猩猩、大鼠和小鼠相应核苷酸序列的同源性分别为98.5%、91.3%、91.0%、88.9%和88.0%.根据获得的cDNA序列,预测已知片段的抗原表位区,并将抗原表位区序列插入pET-44a(+)载体,经转化得到重组菌,经IPTG诱导表达,并通过亲和柱层析,纯化制备GFRα1抗原表位多肽.Western blot检测发现,经诱导表达的GFRα1抗原表位多肽约为18.2 ku,与预测的大小一致.绵羊Gfrα1基因的克隆和表达研究,为进一步制作该基因的多克隆抗体奠定基础,为绵羊SSCs的分子水平鉴定及功能分析提供了研究条件.%In order to clone the DNA sequence of Gfral gene in sheep and deeply research the sheep spermatogonial stem cells (SSCs), in the present study, the partial sheep Gfral cDNA were cloned using RT-PCR and molecular cloning technique. The cloned partial Gfral gene was 1 312 bp in length, including an ORF of 1 311 bp encoding 437 amino acids. The nucleotide sequences of sheep Gfral gene were compared with the counterpart sequences of Bos Taurus , homo sapiens , Pongo abelii , Rattus norvegicus and Mus musculus , and the nucleotide homology was 98. 5% , 91. 3 % , 91.0%, 88. 9% and 88. 0% , respectively. According to the cloned Gfral gene sequence, the epitope was forecasted and then the cDNA was inserted into pET-44a( + ) vector. Subsequently, the recombinant was induced by IPTG. The recombinant epitope polypeptide was purified according to Affinity column chromatography. Western blot analysis indicated that the molecular weight of the expressed epitope polypeptide was the same as predicted size of approximate 18. 2 ku. The data collected

  16. Functional characterization of a monoclonal antibody epitope using a lambda phage display-deep sequencing platform

    Science.gov (United States)

    Domina, Maria; Lanza Cariccio, Veronica; Benfatto, Salvatore; Venza, Mario; Venza, Isabella; Borgogni, Erica; Castellino, Flora; Midiri, Angelina; Galbo, Roberta; Romeo, Letizia; Biondo, Carmelo; Masignani, Vega; Teti, Giuseppe; Felici, Franco; Beninati, Concetta

    2016-01-01

    We have recently described a method, named PROFILER, for the identification of antigenic regions preferentially targeted by polyclonal antibody responses after vaccination. To test the ability of the technique to provide insights into the functional properties of monoclonal antibody (mAb) epitopes, we used here a well-characterized epitope of meningococcal factor H binding protein (fHbp), which is recognized by mAb 12C1. An fHbp library, engineered on a lambda phage vector enabling surface expression of polypeptides of widely different length, was subjected to massive parallel sequencing of the phage inserts after affinity selection with the 12C1 mAb. We detected dozens of unique antibody-selected sequences, the most enriched of which (designated as FrC) could largely recapitulate the ability of fHbp to bind mAb 12C1. Computational analysis of the cumulative enrichment of single amino acids in the antibody-selected fragments identified two overrepresented stretches of residues (H248-K254 and S140-G154), whose presence was subsequently found to be required for binding of FrC to mAb 12C1. Collectively, these results suggest that the PROFILER technology can rapidly and reliably identify, in the context of complex conformational epitopes, discrete “hot spots” with a crucial role in antigen-antibody interactions, thereby providing useful clues for the functional characterization of the epitope. PMID:27530334

  17. Prediction and identification of mouse cytotoxic T lymphocyte epitopes in Ebola virus glycoproteins

    Directory of Open Access Journals (Sweden)

    Wu Shipo

    2012-06-01

    Full Text Available Abstract Background Ebola viruses (EBOVs cause severe hemorrhagic fever with a high mortality rate. At present, there are no licensed vaccines or efficient therapies to combat EBOV infection. Previous studies have shown that both humoral and cellular immune responses are crucial for controlling Ebola infection. CD8+ T cells play an important role in mediating vaccine-induced protective immunity. The objective of this study was to identify H-2d-specific T cell epitopes in EBOV glycoproteins (GPs. Results Computer-assisted algorithms were used to predict H-2d-specific T cell epitopes in two species of EBOV (Sudan and Zaire GP. The predicted peptides were synthesized and identified in BALB/c mice immunized with replication-deficient adenovirus vectors expressing the EBOV GP. Enzyme-linked immunospot assays and intracellular cytokine staining showed that the peptides RPHTPQFLF (Sudan EBOV, GPCAGDFAF and LYDRLASTV (Zaire EBOV could stimulate splenoctyes in immunized mice to produce large amounts of interferon-gamma. Conclusion Three peptides within the GPs of two EBOV strains were identified as T cell epitopes. The identification of these epitopes should facilitate the evaluation of vaccines based on the Ebola virus glycoprotein in a BALB/c mouse model.

  18. 腺病毒载体介导的人分泌型α(1,2)岩藻糖基转移酶的表达降低Galα(1,3)Gal抗原表位水平%Adenovirus-mediated expression of human secretor type a(1,2)fucosyltransferase reduces level of Gala(1,3)Gal epitop

    Institute of Scientific and Technical Information of China (English)

    邢力; 夏国宏; 费俭; 白旭芳; 郭礼和

    2000-01-01

    AIM: To test the potential of human secretor type α(1, 2) fucosyltransferase [Seα(1,2)FT] to downregulate the expression of Galα(1,3)Gal epitope (gal epitope) in cultured cell lines. METHODS: Expression of Seα(1,2) FT was mediated by human adenoviral vector. Flow cytometric analysis was used to compare the expression level of H blood group antigen or gal epitope. MTT was employed to assess the susceptibility of mouse NIH3T3 cells to human natural antibody and complement mediated lysis. RESULTS: A recombinant replication-deficient adenovims (rAdv) containing human Seα( 1,2)FT cDNA (Ad5hSeFT) was designed and successfully constructed. Flow cytometric analysis showed that after mock infection, Ad5null infection, and Ad5hSeFT infection, the mean fluorescence intensity (MFI) values for the binding of Ulex europaeus I (UEA-I) lectin to NIH3T3 cells were 2.3 ± 0.6, 2.1 ± 1.0, and 36.5 ± 5.9, respectively; MFI values for the binding of Griffonia simplicifolia isolectin B4 (GS-IB4) lectin to NIH3T3 cells were 167 ±23, 170 ± 19, and 100 ± 14, respectively; MFI values for the binding of human natural IgG and IgM antibodies to NIH3T3 cells were 31 ± 3, 32 ± 4, and 22 ± 4, respectively. CONCLUSION: H blood group antigen was detected on NIH3T3 cells after Ad5hSeFT infection and resulted in more than 40 % reduction in the level of gal epitope on the cell surface. This reduction increased the resistance of NIH3T3 cells to lysis by normal human serum.%目的:检测人分泌型α(1,2)岩藻糖基转移酶降低Galα(1,3)Gal表位(gal表位)水平.方法:以人腺病毒载体表达人分泌型α(1,2)岩藻糖基转移酶.流式细胞术比较H血型抗原和gal表位的表达水平.MTT法分析小鼠NIH3T3细胞对人天然抗体和补体介导的细胞裂解作用的敏感性.结果:设计并构建了表达人分泌型α(1,2)岩藻糖基转移酶基因的复制缺陷型重组腺病毒载体Ad5hSeFT.流式细胞分析结果表明,NIH3T3细胞及Ad5null

  19. Expression profile of saccharide epitope CaMBr1 in normal and neoplastic tissue from dogs, cats, and rats: implication for the development of human-derived cancer vaccines.

    Science.gov (United States)

    Adobati, E; Zacchetti, A; Perico, M E; Cremonesi, F; Rasi, G; Vallebona, P S; Hagenaars, M; Kuppen, P J; Pastan, I; Panza, L; Russo, G; Colnaghi, M I; Canevari, S

    1999-11-01

    CaMBr1 is a blood group-related tumour-associated antigen, whose pattern of expression provides a therapeutic window for passive or active immunotherapy and points to the promise of a vaccine against carcinomas overexpressing this antigen. In this context, an animal model that closely mimics the human situation would be extremely useful. We, therefore, utilised the murine monoclonal antibody MBr1, which defines CaMBr1, as a useful probe to detect the molecule targeted for vaccine development on canine and feline spontaneous breast and uterus tumours and on their normal counterparts, and on rat normal tissues and carcinoma cell lines. Immunoperoxidase staining of cryostat sections revealed homogeneous CaMBr1 expression only in normal feline uterus and a uterus papilloma, whereas MBr1 reactivity was very weak and heterogeneous in normal (1/3 and 1/3) and tumour (1/10 and 1/6) breast tissues from dogs and cats, respectively. In contrast, the data obtained in rat tissues were reproducible in the strains tested and showed that CaMBr1 was expressed in all epithelial tissues of the digestive tract, although with variable intensities. Monoclonal antibody staining appeared to correspond to membrane-bound structures as well as mucinous secretions. Similarly, secretion products of lactating mammary glands expressed CaMBr1. The spectrum of expression on rat digestive tract was broader than that in humans but the specificity of MBr1 reactivity was confirmed by competition assay with a synthetic tetrasaccharide that mimics the CaMBr1 antigen. On FACS analysis, only one of two clonal derivatives of the rat breast carcinoma line RAMA 25 expressed CaMBr1, and a negative cell subset was evident in repeated experiments. By contrast, both colon carcinoma lines, DHD/K12 and CC531, showed staining with MBr1, albeit at different levels of intensity, and no evidence of a negative subset. The cell line CC531 maintained or even increased CaMBr1 expression levels following transplantation in

  20. Proof of principle for epitope-focused vaccine design

    Science.gov (United States)

    Correia, Bruno E.; Bates, John T.; Loomis, Rebecca J.; Baneyx, Gretchen; Carrico, Chris; Jardine, Joseph G.; Rupert, Peter; Correnti, Colin; Kalyuzhniy, Oleksandr; Vittal, Vinayak; Connell, Mary J.; Stevens, Eric; Schroeter, Alexandria; Chen, Man; MacPherson, Skye; Serra, Andreia M.; Adachi, Yumiko; Holmes, Margaret A.; Li, Yuxing; Klevit, Rachel E.; Graham, Barney S.; Wyatt, Richard T.; Baker, David; Strong, Roland K.; Crowe, James E.; Johnson, Philip R.; Schief, William R.

    2014-03-01

    Vaccines prevent infectious disease largely by inducing protective neutralizing antibodies against vulnerable epitopes. Several major pathogens have resisted traditional vaccine development, although vulnerable epitopes targeted by neutralizing antibodies have been identified for several such cases. Hence, new vaccine design methods to induce epitope-specific neutralizing antibodies are needed. Here we show, with a neutralization epitope from respiratory syncytial virus, that computational protein design can generate small, thermally and conformationally stable protein scaffolds that accurately mimic the viral epitope structure and induce potent neutralizing antibodies. These scaffolds represent promising leads for the research and development of a human respiratory syncytial virus vaccine needed to protect infants, young children and the elderly. More generally, the results provide proof of principle for epitope-focused and scaffold-based vaccine design, and encourage the evaluation and further development of these strategies for a variety of other vaccine targets, including antigenically highly variable pathogens such as human immunodeficiency virus and influenza.

  1. Advances of Bioinformatics Tools Applied in Virus Epitopes Prediction

    Institute of Scientific and Technical Information of China (English)

    Ping Chen; Simon Rayner; Kang-hong Hu

    2011-01-01

    In recent years, the in silico epitopes prediction tools have facilitated the progress of vaccines development significantly and many have been applied to predict epitopes in viruses successfully. Herein, a general overview of different tools currently available, including T cell and B cell epitopes prediction tools, is presented. And the principles of different prediction algorithms are reviewed briefly. Finally, several examples are present to illustrate the application of the prediction tools.

  2. Atomic-level mapping of antibody epitopes on a GPCR.

    Science.gov (United States)

    Paes, Cheryl; Ingalls, Jada; Kampani, Karan; Sulli, Chidananda; Kakkar, Esha; Murray, Meredith; Kotelnikov, Valery; Greene, Tiffani A; Rucker, Joseph B; Doranz, Benjamin J

    2009-05-27

    Epitopes that define the immunodominant regions of conformationally complex integral membrane proteins have been difficult to reliably delineate. Here, a high-throughput approach termed shotgun mutagenesis was used to map the binding epitopes of five different monoclonal antibodies targeting the GPCR CCR5. The amino acids, and in some cases the atoms, that comprise the critical contact points of each epitope were identified, defining the immunodominant structures of this GPCR and their physicochemistry.

  3. New Viral Vector for Superproduction of Epitopes of Vaccine Proteins in Plants

    Science.gov (United States)

    Tyulkina, L.G.; Skurat, E.V.; Frolova, O.Yu.; Komarova, T.V.; Karger, E.M.; Atabekov, I.G.

    2011-01-01

    The novel viral vectors PVX-CP AltMV and PVXdt-CP AltMV are superexpressors of the capsid protein (CP). These viral vectors were constructed on the basis of the potato virus X (PVX) genome andAlternantheramosaic virus (AltMV) CP gene. The expression, based on the hybrid viral vectors, is genetically safe, since the systemic transport and formation of infective viral particles are blocked. CP AltMV can self-assemble into virus-like particles (VLPs) in the absence of genomic RNA. The vectors can be used for the presentation of foreign peptides (including epitopes of human pathogens) on the surface of the VLP. The N-terminal extracellular domain (M2e) of the influenza virus A M2 protein and its truncated variant (ΔM2e) were used as model heterologous peptides for the construction of the chimeric CP AltMV. Chimeric CP AltMV retains its ability to self-assemble into VLP. The epitopes of the M2 influenza virus protein were not eliminated during the process of accumulation, polymerization and purification of chimeric VLP AltMV, providing evidence of the stability of chimeric VLP with C-terminal heterologous epitopes. It appears that VLP produced by the vectors PVX-CP AltMV and PVXdt-CP AltMV can be used in the field of biotechnology for the presentation of the epitopes of vaccine proteins on their surfaces. The chimeric VLP AltMV with the presented foreign epitopes can be used as candidate vaccines. PMID:22649706

  4. Babesia bigemina: identification of B cell epitopes associated with parasitized erythrocytes.

    Science.gov (United States)

    Vidotto, O; McElwain, T F; Machado, R Z; Perryman, L E; Suarez, C E; Palmer, G H

    1995-12-01

    Rhoptries are involved in host cell invasion and rhoptry polypeptides, including the Babesia bigemina rhoptry-associated protein-1 (RAP-1), are targets for protective immune responses. Polyclonal antisera produced against isolated rhoptries is directed predominantly against RAP-1 and reacts with both the merozoite and the membrane of parasitized erythrocytes. To determine whether these B cell epitopes associated with the parasitized erythrocyte are derived from RAP-1 or, alternatively, from previously undetected merozoite polypeptides, monoclonal antibodies (mAbs) were generated from mice immunized with rhoptries isolated from the JG-29 clone of the Mexico strain. The anti-RAP-1 mAbs bound only merozoites in a punctate immunofluorescence pattern. A second group of four mAbs, none of which were reactive with RAP-1, bound the parasitized erythrocyte. Two of these latter mAbs, 64/44.17.3 and 64/05.7.2, reacted only with parasitized erythrocytes that had been permeabilized. MAb 64/44.17.3 bound a 54-kDa merozoite polypeptide while 64/05.7.2 bound a > or = 225-kDa merozoite polypeptide. MAbs 64/32.8.5 and 64/38.5.3 recognized epitopes on 17.5- and 76-kDa polypeptides exposed on the external surface of intact parasitized erythrocytes. The results indicate that the identified RAP-1 epitopes are not associated with the erythrocyte cytoskeleton or membrane and that anti-RAP-1 immunity is most likely generated against the free merozoite. All new mAbs reacted with every B. bigemina strain tested (Mexico, Puerto Rico, St. Croix, Texcoco, Jaboticabal). The conservation of RAP-1 epitopes among these strains supports the continued testing of RAP-1 as a vaccine component. In addition, the identification of epitopes expressed on the surface of erythrocytes infected with all five strains provides new candidate immunogens.

  5. Analysis and application of a neutralizing linear epitope on liable toxin B of enterotoxin Escherichia coli.

    Science.gov (United States)

    Guan, Weikun; Liu, Wenxin; Bao, Jun; Li, Jinping; Yuan, Chaowen; Tang, Jie; Shi, Dongfang

    2015-07-01

    Heat-labile enterotoxin (LT) of enterotoxigenic Escherichia coli (ETEC) is one of the major virulence factors for causing diarrhea in piglets, and LT is a strong immunogen. Thus, LT represents an important target for development of vaccines and diagnostic tests. In this study, bioinformatic tools were used to predict six antigenic B cell epitopes in the B subunit of LT protein (LTB) of ETEC strains. Then, seven antigenic B cell epitopes of LTB were identified by polyclonal antisera (polyclonal antibody (PAb)) using a set of LTB-derived peptides expressed as maltose-binding protein (MBP) fusion protein. In addition, one LTB-specific monoclonal antibody (MAb) was generated and defined its corresponding epitope as mentioned above. This MAb was able to specifically bind with native LT toxin and has no cross-reaction with LT-II (type II heat-labile enterotoxin), Stx1 (Shiga toxin I), Stx2 (Shiga toxin II), STa (heat-stable enterotoxin I), and STb (heat-stable enterotoxin II) toxins. Further, this MAb was able to interrupt LT toxin specific binding to GM1 receptor, indicating that the corresponding epitope is the specific binding region to GM1 receptor. Moreover, in vitro and in vivo assay showed that the MAb was able to neutralize the native LT toxin. Diarrheal suckling pigs challenged with LT-positive ETEC strain recovered when an enema with this purified MAb was administered. This study will provide the foundation for further studies about the interaction between LT toxin and GM1 receptor and about the developing of epitope-based vaccines and specific therapeutic agent.

  6. Construction and infectivity analysis of live attenuated recombinant salmonella expressing HIV-1 447-52D epitope on fimbriae%以沙门氏菌菌毛为载体的重组HIV-1 447-52D表位疫苗的构建和毒力检测

    Institute of Scientific and Technical Information of China (English)

    金刚; 于璐; 岳超; 李海宁; 杨全; 李庆海; 刘树林

    2016-01-01

    Objective To construct recombinant attenuated Salmonella expressing the HIV-1 447-52D epitope on the thin aggregative fimbriae and to analyze the infectivity status of this recombinant strain.Methods The recombinant attenuated Salmonella ST-44752D was constructed by using pHSG415 gene replacement system to replace the wild-type agfA gene with the chimeric agfA:44752D gene and then verified by polymeruse chain reaction(PCR) and sequencing.The expression of the recombinant thin aggregative fimbriae was analyzed by Western blotting.Congo red binding test was performed to examine the adsorbability of fimbriae.50% lethal dose (LD50) and organ distribution status of the 447-52D strain were determined in mice.Results The 447-52D expressing recombinant strain was successfully constructed and confirmed by DNA sequencing.Compared with wild-type ST,recombinant ST-44752D strain showed similar expression level of the thin aggregative fimbriae AgfA,and similar binding activity to Congo red reagent.The LD50 value of ST-44752D strain in mice was 1.45 × 109 CFU.After infection,ST-44752D strain could distribute effectively in spleen and lymph node,similar to wild-type ST.Conclusion The recombinant attenuated Salmonella typhimurium ST-44752D harboring HIV-1 447-52D epitope on fimbriae was successfully constructed.The 447-52D epitope within AgfA did not influence AgfA expression,fimbrial adsorbability,bacterial LDS0 value or organ distribution in mice.%目的 构建菌毛上表达HIV-1 447-52D表位的减毒Salmonella typhimurium ST14028-3b aroA-44752D,并对其毒力进行检测.方法 利用同源重组技术,将447-52D表位整合至沙门氏菌细聚合菌毛上,Western blot检测其表达情况,通过刚果红结合实验分析外源表位对菌毛吸附能力的影响.感染动物后检测重组菌的毒力及其在小鼠体内的分布.结果 重组菌的嵌合基因片段测序结果和嵌合蛋白的表达分析表明,重组菌菌毛上成功表达外源447-52D表位.重

  7. Biochemical, biophysical and IgE-epitope characterization of the wheat food allergen, Tri a 37.

    Directory of Open Access Journals (Sweden)

    Sandra Pahr

    Full Text Available Wheat is an important staple food and potent allergen source. Recently, we isolated a cDNA coding for wheat alpha-purothionin which is recognized by wheat food allergic patients at risk for severe wheat-induced allergy. The purpose of the present study was the biochemical, biophysical and IgE epitope characterization of recombinant alpha-purothionin. Synthetic genes coding for alpha-purothionin were expressed in a prokaryotic system using Escherichia coli and in a eukaryotic expression system based on baculovirus-infected Sf9-insect cells. Recombinant proteins were purified and characterized by SDS-PAGE, mass spectrometry, circular dichroism, chemical cross-linking and size exclusion chromatography. Five overlapping peptide were synthesized for epitope mapping. Alpha-purothionin-specific rabbit antibodies were raised to perform IgE-inhibition experiments and to study the resistance to digestion. The IgE reactivity of the proteins and peptides from ten wheat food allergic patients was studied in non-denaturing RAST-based binding assays. Alpha-purothionin was expressed in the prokaryotic (EcTri a 37 and in the eukaryotic system (BvTri a 37 as a soluble and monomeric protein. However, circular dichroism analysis revealed that EcTri a 37 was unfolded whereas BvTri a 37 was a folded protein. Both proteins showed comparable IgE-reactivity and the epitope mapping revealed the presence of sequential IgE epitopes in the N-terminal basic thionin domain (peptide1:KSCCRSTLGRNCYNLCRARGAQKLCAGVCR and in the C-terminal acidic extension domain (peptide3:KGFPKLALESNSDEPDTIEYCNLGCRSSVC, peptide4:CNLGCRSSVCDYMVNAAADDEEMKLYVEN. Natural Tri a 37 was digested under gastric conditions but resistant to duodenal digestion. Immunization with EcTri a 37 induced IgG antibodies which recognized similar epitopes as IgE antibodies from allergic patients and inhibited allergic patients' IgE binding. Reactivity to Tri a 37 does not require a folded protein and the presence of

  8. HIV-1 vaccine-induced T-cell responses cluster in epitope hotspots that differ from those induced in natural infection with HIV-1.

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    Tomer Hertz

    Full Text Available Several recent large clinical trials evaluated HIV vaccine candidates that were based on recombinant adenovirus serotype 5 (rAd-5 vectors expressing HIV-derived antigens. These vaccines primarily elicited T-cell responses, which are known to be critical for controlling HIV infection. In the current study, we present a meta-analysis of epitope mapping data from 177 participants in three clinical trials that tested two different HIV vaccines: MRKAd-5 HIV and VRC-HIVAD014-00VP. We characterized the population-level epitope responses in these trials by generating population-based epitope maps, and also designed such maps using a large cohort of 372 naturally infected individuals. We used these maps to address several questions: (1 Are vaccine-induced responses randomly distributed across vaccine inserts, or do they cluster into immunodominant epitope hotspots? (2 Are the immunodominance patterns observed for these two vaccines in three vaccine trials different from one another? (3 Do vaccine-induced hotspots overlap with epitope hotspots induced by chronic natural infection with HIV-1? (4 Do immunodominant hotspots target evolutionarily conserved regions of the HIV genome? (5 Can epitope prediction methods be used to identify these hotspots? We found that vaccine responses clustered into epitope hotspots in all three vaccine trials and some of these hotspots were not observed in chronic natural infection. We also found significant differences between the immunodominance patterns generated in each trial, even comparing two trials that tested the same vaccine in different populations. Some of the vaccine-induced immunodominant hotspots were located in highly variable regions of the HIV genome, and this was more evident for the MRKAd-5 HIV vaccine. Finally, we found that epitope prediction methods can partially predict the location of vaccine-induced epitope hotspots. Our findings have implications for vaccine design and suggest a framework by which

  9. Relation Analysis of Synovial Anti-Citrullinated Epitope Peptide Expression and Peptidyl Arginine Deiminase 4 Gene in the Rheumatoid Arthritis Patient%类风湿关节炎患者滑膜抗环瓜氨酸肽表位表达与肽酰基精氨酸脱亚氨酶4基因的相关性分析

    Institute of Scientific and Technical Information of China (English)

    沈小辉; 喻伟; 杨菲

    2016-01-01

    Objective To investigate the relation of synovial anti-citrullinated epitope peptide(CCP)expression and peptidyl arginine deiminase 4 (PADI4)gene in the rheumatoid arthritis patient.Methods From February 2013 to 2015 in Dongfeng General Hospital Affiliated to Hubei University,selected 110 patients with rheumatoid arthritis as the observation group, and selected 110 healthy people at the same period in this hospital for medical examination as the control group,both groups were given the synovial CCP expression positive rate and PADI4 gene expression detecting and correlation analysis.Results The synovial anti-CCP expression positive rates in the observation group and the control group were 70.9% and 9.1% re-spectively,and the observation group were significantly higher than the control group (P<0.05).The PADI4-104 genotype expression frequency compared between the observation group and control group,the difference were statistically significant (P<0.05).The G/G expression was more in the observation group,while the control group was more wit the C/G expres-sion.Pearson correlation analysis showed that in the observation group,synovial CCP positive expression were significant correlated to the expression of genotype PADI4-104 (r=0.344,P<0.05).Logistic regression analysis showed PADI4-104 genotype frequency were the main factors for the synovial CCP epitope expression (P<0.05).Conclusion The rheumatoid arthritis was more with synovial anti-CCP positive expression.There were also PADI4 polymorphism disorder expression, and clear correlation between the two.Thus impact the incidence of rheumatoid arthritis.%目的探讨类风湿关节炎患者滑膜抗环瓜氨酸肽(CCP)表位表达与肽酰基精氨酸脱亚氨酶4(PADI4)基因的相关性。方法2013年2月~2015年选择在湖北医药学院附属东风医院风湿免疫科住院的类风湿关节炎患者110例作为观察组,同期选择在该院进行体检的健康者110例作为对照

  10. Epitope DNA vaccines against tuberculosis: spacers and ubiquitin modulates cellular immune responses elicited by epitope DNA vaccine

    Institute of Scientific and Technical Information of China (English)

    Wang QM; Sun SH; Hu ZL; Zhou FJ; Yin M; Xiao CJ; Zhang JC

    2005-01-01

    Cell-mediated immune responses are crucial in the protection against tuberculosis. In this study, we constructed epitope DNA vaccines (p3-M-38) encoding cytotoxic T lymphocyte (CTL) epitopes of MPT64 and 38 kDa proteins of Mycobacterium tuberculosis. In order to observe the influence of spacer sequence (Ala-Ala-Tyr) or ubiquitin (UbGR) on the efficacy of the two CTL epitopes, we also constructed DNA vaccines, p3-M-S(spacer)-38, p3-Ub (UbGR)-M-S-38 and p3-Ub-M-38. The immune responses elicited by the four DNA vaccines were tested in C57BL/6 (H-2b) mice. The cytotoxicity of T cells was detected by LDH-release method and by enzyme-linked immunospot assay for epitope-specific cells secreting interferon-gamma. The results showed that DNA immunization with p3-M-38 vaccine could induce epitope-specific CD8+ CTL response and that the spacer sequence (AAY) only enhanced M epitope presentation. The protein-targeting sequence (UbGR) enhanced the immunogenicity of the two epitopes. The finding that defined spacer sequences at C-terminus and protein-targeting degradation modulated the immune response of epitope string DNA vaccines will be of importance for the further development of multi-epitope DNA vaccines against tuberculosis.

  11. Identification and characterization of a virus-specific continuous B-cell epitope on the PrM/M protein of Japanese Encephalitis Virus: potential application in the detection of antibodies to distinguish Japanese Encephalitis Virus infection from West Nile Virus and Dengue Virus infections

    Directory of Open Access Journals (Sweden)

    Liu Wen-Xin

    2010-09-01

    Full Text Available Abstract Background Differential diagnose of Japanese encephalitis virus (JEV infection from other flavivirus especially West Nile virus (WNV and Dengue virus (DV infection was greatly hindered for the serological cross-reactive. Virus specific epitopes could benefit for developing JEV specific antibodies detection methods. To identify the JEV specific epitopes, we fully mapped and characterized the continuous B-cell epitope of the PrM/M protein of JEV. Results To map the epitopes on the PrM/M protein, we designed a set of 20 partially overlapping fragments spanning the whole PrM, fused them with GST, and expressed them in an expression vector. Linear epitope M14 (105VNKKEAWLDSTKATRY120 was detected by enzyme-linked immunosorbent assay (ELISA. By removing amino acid residues individually from the carboxy and amino terminal of peptide M14, we confirmed that the minimal unit of the linear epitope of PrM/M was M14-13 (108KEAWLDSTKAT118. This epitope was highly conserved across different JEV strains. Moreover, this epitope did not cross-react with WNV-positive and DENV-positive sera. Conclusion Epitope M14-13 was a JEV specific lineal B-cell epitpe. The results may provide a useful basis for the development of epitope-based virus specific diagnostic clinical techniques.

  12. Identification and characterisation of T-cell epitopes for incorporation into dendritic cell-delivered Listeria vaccines.

    Science.gov (United States)

    Calderon-Gonzalez, Ricardo; Tobes, Raquel; Pareja, Eduardo; Frande-Cabanes, Elisabet; Petrovsky, Nikolai; Alvarez-Dominguez, Carmen

    2015-09-01

    Dendritic cells loaded with antigenic peptides, because of their safety and robust immune stimulation, would be ideal for induction of immunity to protect against listeriosis. However, there is no currently accepted method to predict which peptides derived from the Listeria proteome might confer protection. While elution of peptides from MHC molecules after Listeria infection yields high-affinity immune-dominant epitopes, these individual epitopes did not reliably confer Listeria protection. Instead we applied bioinformatic predictions of MHC class I and II epitopes to generate antigenic peptides that were then formulated with Advax™, a novel polysaccharide particulate adjuvant able to enhance cross-presentation prior to being screened for their ability to induce protective T-cell responses. A combination of at least four intermediate strength MHC-I binding epitopes and one weak MHC-II binding epitope when expressed in a single peptide sequence and formulated with Advax adjuvant induced a potent T-cell response and high TNF-α and IL-12 production by dendritic cells resulting in robust listeriosis protection in susceptible mice. This T-cell vaccine approach might be useful for the design of vaccines to protect against listeriosis or other intracellular infections.

  13. Stable, conserved outer membrane epitope of strains of Haemophilus influenzae biogroup aegyptius associated with Brazilian purpuric fever.

    Science.gov (United States)

    Lesse, A J; Gheesling, L L; Bittner, W E; Myers, S D; Carlone, G M

    1992-04-01

    Brazilian purpuric fever is a rapidly fatal childhood disease associated with a clonal strain of Haemophilus influenzae biogroup aegyptius. We describe a conserved, surface-exposed epitope present on 95% of H. influenzae biogroup aegyptius isolates that are associated with Brazilian purpuric fever. This epitope, defined by reaction with the monoclonal antibody 8G3, is on or associated with the 48-kDa heat-modifiable P1 protein. The epitope is absent on strains of H. influenzae biogroup aegyptius that are not associated with Brazilian purpuric fever but is present on one strain of H. influenzae biotype II. None of 81 other Haemophilus strains tested reacted with 8G3. The sensitivity and specificity of the 8G3 monoclonal antibody in detecting Brazilian case-clone strains of H. influenzae biogroup aegyptius associated with Brazilian purpuric fever are 95 and 99%, respectively. Immunoelectron microscopy revealed that the epitope is surface exposed, and N-terminal amino acid sequencing of an 8G3-reactive P1 protein from a strain of H. influenzae biogroup aegyptius showed 100% correlation with the published N-terminal amino acid sequence of a P1 protein of H. influenzae type b. The virulence of the organism in an infant rat model of bacteremia was not dependent on the expression of this epitope.

  14. Isolating subpopulations of human epidermal basal cells based on polyclonal serum against trypsin-resistant CSPG4 epitopes

    DEFF Research Database (Denmark)

    Gunnarsson, Anders Patrik Alexander; Christensen, Rikke; Prætorius, Jeppe

    2016-01-01

    unable to study these keratinocytes isolated directly from skin samples by flow cytometry. By choosing epitopes of CSPG4 relatively close to the cell membrane we were able to generate a polyclonal antibody that successfully detects CSPG4 on keratinocytes after trypsinization. Although CSPG4-positive......Chondroitin sulfate proteoglycan 4 (CSPG4) is highly expressed by human epidermal keratinocytes located at the tip of the dermal papilla where keratinocytes show characteristics of stem cells. However, since available antibodies to CSPG4 are directed against trypsin-sensitive epitopes we have been...... that CSPG4-positive basal cell keratinocytes are distinct from CSPG4-negative basal cell keratinocytes. Our study demonstrates that it is possible to generate antibodies against trypsin-resistant epitopes of CSPG4. Our study also documents a marked change in behaviour upon cell culturing and challenges...

  15. Conservation and diversity of influenza A H1N1 HLA-restricted T cell epitope candidates for epitope-based vaccines.

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    Paul Thiamjoo Tan

    Full Text Available BACKGROUND: The immune-related evolution of influenza viruses is exceedingly complex and current vaccines against influenza must be reformulated for each influenza season because of the high degree of antigenic drift among circulating influenza strains. Delay in vaccine production is a serious problem in responding to a pandemic situation, such as that of the current H1N1 strain. Immune escape is generally attributed to reduced antibody recognition of the viral hemagglutinin and neuraminidase proteins whose rate of mutation is much greater than that of the internal non-structural proteins. As a possible alternative, vaccines directed at T cell epitope domains of internal influenza proteins, that are less susceptible to antigenic variation, have been investigated. METHODOLOGY/PRINCIPAL FINDINGS: HLA transgenic mouse strains expressing HLA class I A*0201, A*2402, and B*0702, and class II DRB1*1501, DRB1*0301 and DRB1*0401 were immunized with 196 influenza H1N1 peptides that contained residues of highly conserved proteome sequences of the human H1N1, H3N2, H1N2, H5N1, and avian influenza A strains. Fifty-four (54 peptides that elicited 63 HLA-restricted peptide-specific T cell epitope responses were identified by IFN-gamma ELISpot assay. The 54 peptides were compared to the 2007-2009 human H1N1 sequences for selection of sequences in the design of a new candidate H1N1 vaccine, specifically targeted to highly-conserved HLA-restricted T cell epitopes. CONCLUSIONS/SIGNIFICANCE: Seventeen (17 T cell epitopes in PB1, PB2, and M1 were selected as vaccine targets based on sequence conservation over the past 30 years, high functional avidity, non-identity to human peptides, clustered localization, and promiscuity to multiple HLA alleles. These candidate vaccine antigen sequences may be applicable to any avian or human influenza A virus.

  16. Construction and characterization of a hexon-chimeric human adenovirus type 3 vector expressing one major epitope of dengue virus type 1%1型登革病毒抗原表位嵌合人3型腺病毒六邻体重组病毒的构建及免疫学鉴定

    Institute of Scientific and Technical Information of China (English)

    招穗珊; 周志超; 李潇; 樊晔; 廖小红; 周荣; 苏晓波

    2015-01-01

    Objective To construct a recombinant human adenovirus type 3 ( HAd3 ) vector ex-pressing one major epitope of dengue virus type 1.Methods The gene encoding the envelope protein (304-314 aa) of dengue virus type 1 was inserted into the hypervariable region 1 ( HVR1 ) of HAd3 hexon by using overlap PCR.The recombinant gene was cloned into the shuttle plasmid, then linearized with AsisⅠrestriction enzyme and co-transformed into Escherichia coli BJ5183 strains with the digested backbone plas-mid for homologous recombination.The recombinant plasmid pBRAdΔE3GFP-DENV1 was transfected into AD293 cells to rescue recombinant adenovirus strains (rAdΔE3GFP-DENV1).ELISA and Western blot as-say were performed to evaluate the humoral responses induced in BALB/c mice after the immunization with rAdΔE3GFP-DENV1 strains.Results The recombinant adenovirus strains were successfully rescued. ELISA and Western blot assay showed that the antibodies in serum sample could recognize dengue virus type 1 strains.Conclusion The recombinant adenovirus strains expressing the epitope of dengue virus type 1 were successfully constructed.This study provided evidence for the development of multivalent vaccines against dengue virus.%目的:构建六邻体嵌入1型登革病毒( DENV1)抗原表位的人3型重组腺病毒,鉴定其抗原性。方法以人3型腺病毒骨架质粒pBRAdΔE3GFP为模板,overlap PCR在六邻体高变区HVR1插入DENV1的抗原表位,突变的六邻体片段克隆到穿梭载体,酶切后与线性化的3型腺病毒骨架质粒pBRAdΔE3GFP在大肠杆菌BJ5183同源重组,获得阳性重组腺病毒质粒pBRAdΔE3GFP-DENV1。线性化后转染AD293细胞拯救重组腺病毒rAdΔE3GFP-DENV1并大量培养。纯化后腺病毒免疫BALB/c小鼠,通过ELISA和Western blot检测小鼠的体液免疫应答。结果在人3型腺病毒六邻体成功插入DENV1抗原表位并包装出重组腺病毒,ELISA和Western blot结果显示小鼠免

  17. A xylogalacturonan epitope is specifically associated with plant cell detachment

    DEFF Research Database (Denmark)

    Willats, William George Tycho; McCartney, L.; Steele-King, C.G.;

    2004-01-01

    A monoclonal antibody (LM8) was generated with specificity for xyloglacturonan (XGA) isolated from pea (Pisum sativum L.) testae. Characterization of the LM8 epitope indicates that it is a region of XGA that is highly substituted with xylose. Immunocytochemical analysis indicates that this epitop...... that is specifically associated with a plant cell separation process that results in complete cell detachment....

  18. Distinct activation phenotype of a highly conserved novel HLA-B57-restricted epitope during dengue virus infection.

    Science.gov (United States)

    Townsley, Elizabeth; Woda, Marcia; Thomas, Stephen J; Kalayanarooj, Siripen; Gibbons, Robert V; Nisalak, Ananda; Srikiatkhachorn, Anon; Green, Sharone; Stephens, Henry A F; Rothman, Alan L; Mathew, Anuja

    2014-01-01

    Variation in the sequence of T-cell epitopes between dengue virus (DENV) serotypes is believed to alter memory T-cell responses during second heterologous infections. We identified a highly conserved, novel, HLA-B57-restricted epitope on the DENV NS1 protein. We predicted higher frequencies of B57-NS1(26-34) -specific CD8(+) T cells in peripheral blood mononuclear cells from individuals undergoing secondary rather than primary DENV infection. However, high tetramer-positive T-cell frequencies during acute infection were seen in only one of nine subjects with secondary infection. B57-NS1(26-34) -specific and other DENV epitope-specific CD8(+) T cells, as well as total CD8(+) T cells, expressed an activated phenotype (CD69(+) and/or CD38(+)) during acute infection. In contrast, expression of CD71 was largely limited to DENV epitope-specific CD8(+) T cells. In vitro stimulation of cell lines indicated that CD71 expression was differentially sensitive to stimulation by homologous and heterologous variant peptides. CD71 may represent a useful marker of antigen-specific T-cell activation.

  19. Epitope mapping of a monoclonal antibody against the Gp85 of avian leukosis virus subgroup J.

    Science.gov (United States)

    Sun, Miao; Yu, Duo; Mo, Hongfei; Cao, Hong; Chen, Chen; Chen, Fuyong

    2012-06-01

    Avian leukosis virus subgroup J poses a great threat to the poultry industry in China. To reduce the economic losses, a quick method for detection of ALV-J antigen is required for diagnosis and identification of the congenitally transmitting hens. In this study, we report the production and evaluation of one monoclonal antibody (MAb) suitable for achieving these goals. The gp85 gene of avian leukosis virus subgroup J CAUHM01 China isolates was subcloned into the expression vectors pGEX-6p-1 and pET28a and successfully expressed in E. coli. After immunizing BALB/c mice with recombinant His-Jgp85 protein, splenic cells from immunized mice were fused with SP2/0 myeloma cells to produce hybridomas. We isolated and characterized one ALV-J gp85-specific MAb by determining its titer, affinity and IgG subclass. In addition, we performed epitope mapping and determined the epitope for the MAb 1E3 to be 81-92 aa of ALV-J gp85 protein (LPWDPQELDILG). Bioinformatics analysis and IFA studies revealed that this epitope is conserved among all ALV-J isolates and that this antibody could serve as a useful reagent for ALV-J detection and diagnosis.

  20. Small-fragment genomic libraries for the display of putative epitopes from clinically significant pathogens.

    Science.gov (United States)

    Henics, T; Winkler, B; Pfeifer, U; Gill, S R; Buschle, M; von Gabain, A; Meinke, A L

    2003-07-01

    Taking advantage of whole genome sequences of bacterial pathogens in many thriving diseases with global impact, we developed a comprehensive screening procedure for the identification of putative vaccine candidate antigens. Importantly, this procedure relies on highly representative small-fragment genomic libraries that are expressed to display frame-selected epitope-size peptides on a bacterial cell surface and to interact directly with carefully selected disease-relevant high-titer sera. Here we describe the generation of small-fragment genomic libraries of Gram-positive and Gram-negative clinically significant pathogens, including Staphylococcus aureus and Staphylococcus epidermidis, Streptococcus pyogenes, Streptococcus agalactiae, and Streptococcus pneumoniae, Enterococcus faecalis, Helicobacter pylori, Chlamydia pneumoniae, the enterotoxigenic Escherichia coli, and Campylobacter jejuni. Large-scale sequencing revealed that the libraries, which provide an average of 20-fold coverage, were random and, as demonstrated with two S. aureus libraries, highly representative. Consistent with the comprehensive nature of this approach is the identification of epitopes that reside in both annotated and putatively novel open reading frames. The use of these libraries therefore allows for the rapid and direct identification of immunogenic epitopes with no apparent bias or difficulty that often associate with conventional expression methods.

  1. Biovar-specific epitopes of the urease enzyme of Ureaplasma urealyticum.

    Science.gov (United States)

    MacKenzie, C R; Henrich, B; Hadding, U

    1996-11-01

    The importance of Ureaplasma urealyticum as a pathogen in premature neonates and patients with a profound defect in humoral immunity has, over the last few years, become well recognised. U. urealyticum is unique amongst the Mycoplasmataceae for its use of urea metabolism as an essential source of energy. The urease enzyme responsible for this is, therefore, of prime importance and any variability in expression of this enzyme may play a role in virulence of the organism. U. urealyticum is divided into 14 serovars comprising two biovars -- the parvo-biovar and T960-biovar. In this study monoclonal antibodies (MAbs) were produced against the urease enzyme. Two distinct epitopes of the 72-kDa alpha-subunit were recognised by three different MAbs. Under denaturing conditions both epitopes were shown to be specific for the parvo-biovar.

  2. Analysis of cytotoxic T cell epitopes in relation to cancer

    DEFF Research Database (Denmark)

    Stranzl, Thomas

    kill the infected cells. The focus of my PhD project has been on improving a method for CTL epitope pathway prediction, on analyzing the epitope density in the alternative cancer exome, and on a study investigating minor histocompatibility antigens (mHags) associated with leukemia. Part I......CTL methods, the experimental effort to identify 90% of new epitopes can be reduced by 15% and 40%, respectively. Part III reports the results of an analysis investigating how the alternatively spliced cancer exome differs from the exome of normal tissue in terms of containing predicted MHC class I binding...... epitopes. We show that peptides unique to cancer splice variants comprise significantly fewer predicted HLA class I epitopes than peptides unique to spliced transcripts in normal tissue. We furthermore find that hydrophilic amino acids are significantly enriched in the unique carcinoma sequences, which...

  3. Autoantibody recognition mechanisms of p53 epitopes

    Science.gov (United States)

    Phillips, J. C.

    2016-06-01

    There is an urgent need for economical blood based, noninvasive molecular biomarkers to assist in the detection and diagnosis of cancers in a cost-effective manner at an early stage, when curative interventions are still possible. Serum autoantibodies are attractive biomarkers for early cancer detection, but their development has been hindered by the punctuated genetic nature of the ten million known cancer mutations. A landmark study of 50,000 patients (Pedersen et al., 2013) showed that a few p53 15-mer epitopes are much more sensitive colon cancer biomarkers than p53, which in turn is a more sensitive cancer biomarker than any other protein. The function of p53 as a nearly universal "tumor suppressor" is well established, because of its strong immunogenicity in terms of not only antibody recruitment, but also stimulation of autoantibodies. Here we examine dimensionally compressed bioinformatic fractal scaling analysis for identifying the few sensitive epitopes from the p53 amino acid sequence, and show how it could be used for early cancer detection (ECD). We trim 15-mers to 7-mers, and identify specific 7-mers from other species that could be more sensitive to aggressive human cancers, such as liver cancer. Our results could provide a roadmap for ECD.

  4. Broad epitope coverage of a human in vitro antibody library

    Science.gov (United States)

    Sivasubramanian, Arvind; Lynaugh, Heather; Yu, Yao; Miles, Adam; Eckman, Josh; Schutz, Kevin; Piffath, Crystal; Boland, Nadthakarn; Durand, Stéphanie; Boland, Todd; Vásquez, Maximiliano; Xu, Yingda; Abdiche, Yasmina

    2017-01-01

    ABSTRACT Successful discovery of therapeutic antibodies hinges on the identification of appropriate affinity binders targeting a diversity of molecular epitopes presented by the antigen. Antibody campaigns that yield such broad “epitope coverage” increase the likelihood of identifying candidates with the desired biological functions. Accordingly, epitope binning assays are employed in the early discovery stages to partition antibodies into epitope families or “bins” and prioritize leads for further characterization and optimization. The collaborative program described here, which used hen egg white lysozyme (HEL) as a model antigen, combined 3 key capabilities: 1) access to a diverse panel of antibodies selected from a human in vitro antibody library; 2) application of state-of-the-art high-throughput epitope binning; and 3) analysis and interpretation of the epitope binning data with reference to an exhaustive set of published antibody:HEL co-crystal structures. Binning experiments on a large merged panel of antibodies containing clones from the library and the literature revealed that the inferred epitopes for the library clones overlapped with, and extended beyond, the known structural epitopes. Our analysis revealed that nearly the entire solvent-exposed surface of HEL is antigenic, as has been proposed for protein antigens in general. The data further demonstrated that synthetic antibody repertoires provide as wide epitope coverage as those obtained from animal immunizations. The work highlights molecular insights contributed by increasingly higher-throughput binning methods and their broad utility to guide the discovery of therapeutic antibodies representing a diverse set of functional epitopes. PMID:27748644

  5. B Epitope Multiplicity and B/T Epitope Orientation Influence Immunogenicity of Foot-and-Mouth Disease Peptide Vaccines

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    Esther Blanco

    2013-01-01

    Full Text Available Synthetic peptides incorporating protective B- and T-cell epitopes are candidates for new safer foot-and-mouth disease (FMD vaccines. We have reported that dendrimeric peptides including four copies of a B-cell epitope (VP1 136 to 154 linked to a T-cell epitope (3A 21 to 35 of FMD virus (FMDV elicit potent B- and T-cell specific responses and confer protection to viral challenge, while juxtaposition of these epitopes in a linear peptide induces less efficient responses. To assess the relevance of B-cell epitope multivalency, dendrimers bearing two (B2T or four (B4T copies of the B-cell epitope from type O FMDV (a widespread circulating serotype were tested in CD1 mice and showed that multivalency is advantageous over simple B-T-epitope juxtaposition, resulting in efficient induction of neutralizing antibodies and optimal release of IFNγ. Interestingly, the bivalent B2T construction elicited similar or even better B- and T-cell specific responses than tetravalent B4T. In addition, the presence of the T-cell epitope and its orientation were shown to be critical for the immunogenicity of the linear juxtaposed monovalent peptides analyzed in parallel. Taken together, our results provide useful insights for a more accurate design of FMD subunit vaccines.

  6. EPITOPE MAPPING OF SCLC-CLUSTER-2 MABS AND GENERATION OF ANTIBODIES DIRECTED AGAINST NEW EGP-2 EPITOPES

    NARCIS (Netherlands)

    HELFRICH, W; KONING, PW; THE, TH; DELEIJ, L

    1994-01-01

    Western blot analysis proved that all cluster-2 MAbs recognize identical or overlapping disulfide-bond-dependent epitopes, indicating the presence of a disulfide-bond-stabilized EGP-2 domain carrying highly immunodominant non-linear epitopes. The apparent immunodominance of this domain makes it diff

  7. Immunization against a saccharide epitope accelerates clearance of experimental gonococcal infection.

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    Sunita Gulati

    Full Text Available The emergence of ceftriaxone-resistant strains of Neisseria gonorrhoeae may herald an era of untreatable gonorrhea. Vaccines against this infection are urgently needed. The 2C7 epitope is a conserved oligosaccharide (OS structure, a part of lipooligosaccharide (LOS on N gonorrhoeae. The epitope is expressed by 94% of gonococci that reside in the human genital tract (in vivo and by 95% of first passaged isolates. Absence of the 2C7 epitope shortens the time of gonococcal carriage in a mouse model of genital infection. To circumvent the limitations of saccharide immunogens in producing long lived immune responses, previously we developed a peptide mimic (called PEP1 as an immunologic surrogate of the 2C7-OS epitope and reconfigured it into a multi-antigenic peptide, (MAP1. To test vaccine efficacy of MAP1, female BALB/c mice were passively immunized with a complement-dependent bactericidal monoclonal antibody specific for the 2C7 epitope or were actively immunized with MAP1. Mice immunized with MAP1 developed a TH1-biased anti-LOS IgG antibody response that was also bactericidal. Length of carriage was shortened in immune mice; clearance occurred in 4 days in mice passively administered 2C7 antibody vs. 6 days in mice administered control IgG3λ mAb in one experiment (p = 0.03 and 6 vs. 9 days in a replicate experiment (p = 0.008. Mice vaccinated with MAP1 cleared infection in 5 days vs. 9 days in mice immunized with control peptide (p = 0.0001 and p = 0.0002, respectively in two replicate experiments. Bacterial burden was lower over the course of infection in passively immunized vs. control mice in both experiments (p = 0.008 and p = 0.0005; burdens were also lower in MAP1 immunized mice vs. controls (p<0.0001 and were inversely related to vaccine antibodies induced in the vagina (p = 0.043. The OS epitope defined by mAb 2C7 may represent an effective vaccine target against gonorrhea, which is rapidly becoming

  8. Identification of Candidate Tolerogenic CD8+ T Cell Epitopes for Therapy of Type 1 Diabetes in the NOD Mouse Model

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    Cailin Yu

    2016-01-01

    Full Text Available Type 1 diabetes is an autoimmune disease in which insulin-producing pancreatic islet β cells are the target of self-reactive B and T cells. T cells reactive with epitopes derived from insulin and/or IGRP are critical for the initiation and maintenance of disease, but T cells reactive with other islet antigens likely have an essential role in disease progression. We sought to identify candidate CD8+ T cell epitopes that are pathogenic in type 1 diabetes. Proteins that elicit autoantibodies in human type 1 diabetes were analyzed by predictive algorithms for candidate epitopes. Using several different tolerizing regimes using synthetic peptides, two new predicted tolerogenic CD8+ T cell epitopes were identified in the murine homolog of the major human islet autoantigen zinc transporter ZnT8 (aa 158–166 and 282–290 and one in a non-β cell protein, dopamine β-hydroxylase (aa 233–241. Tolerizing vaccination of NOD mice with a cDNA plasmid expressing full-length proinsulin prevented diabetes, whereas plasmids encoding ZnT8 and DβH did not. However, tolerizing vaccination of NOD mice with the proinsulin plasmid in combination with plasmids expressing ZnT8 and DβH decreased insulitis and enhanced prevention of disease compared to vaccination with the plasmid encoding proinsulin alone.

  9. B Cell Epitope-Based Vaccination Therapy

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    Yoshie Kametani

    2015-08-01

    Full Text Available Currently, many peptide vaccines are undergoing clinical studies. Most of these vaccines were developed to activate cytotoxic T cells; however, the response is not robust. Unlike vaccines, anti-cancer antibodies based on passive immunity have been approved as a standard treatment. Since passive immunity is more effective in tumor treatment, the evidence suggests that limited B cell epitope-based peptide vaccines may have similar activity. Nevertheless, such peptide vaccines have not been intensively developed primarily because humoral immunity is thought to be preferable to cancer progression. B cells secrete cytokines, which suppress immune functions. This review discusses the possibility of therapeutic antibody induction by a peptide vaccine and the role of active and passive B cell immunity in cancer patients. We also discuss the use of humanized mice as a pre-clinical model. The necessity of a better understanding of the activity of B cells in cancer is also discussed.

  10. Hepatitis C virus (HCV infection may elicit neutralizing antibodies targeting epitopes conserved in all viral genotypes.

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    Nicasio Mancini

    Full Text Available Anti-hepatitis C virus (HCV cross-neutralizing human monoclonal antibodies, directed against conserved epitopes on surface E2 glycoprotein, are central tools for understanding virus-host interplay, and for planning strategies for prevention and treatment of this infection. Recently, we developed a research aimed at identifying these antibody specificities. The characteristics of one of these antibodies (Fab e20 were addressed in this study. Firstly, using immunofluorescence and FACS analysis of cells expressing envelope HCV glycoproteins, Fab e20 was able to recognize all HCV genotypes. Secondly, competition assays with a panel of mouse and rat monoclonals, and alanine scanning mutagenesis analyses located the e20 epitope within the CD81 binding site, documenting that three highly conserved HCV/E2 residues (W529, G530 and D535 are critical for e20 binding. Finally, a strong neutralizing activity against HCV pseudoparticles (HCVpp incorporating envelope glycoproteins of genotypes 1a, 1b, 2a, 2b and 4, and against the cell culture-grown (HCVcc JFH1 strain, was observed. The data highlight that neutralizing antibodies against HCV epitopes present in all HCV genotypes are elicited during natural infection. Their availability may open new avenues to the understanding of HCV persistence and to the development of strategies for the immune control of this infection.

  11. Epitope-Specific Vaccination Limits Clonal Expansion of Heterologous Naive T Cells during Viral Challenge

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    Lexus R. Johnson

    2016-10-01

    Full Text Available Despite robust secondary T cell expansion primed by vaccination, the impact on primary immune responses to heterotypic antigens remains undefined. Here we show that secondary expansion of epitope-specific memory CD8+ T cells primed by prior infection with recombinant pathogens limits the primary expansion of naive CD8+ T cells with specificity to new heterologous antigens, dampening protective immunity against subsequent pathogen challenge. The degree of naive T cell repression directly paralleled the magnitude of the recall response. Suppressed primary T cell priming reflects competition for antigen accessibility, since clonal expansion was not inhibited if the primary and secondary epitopes were expressed on different dendritic cells. Interestingly, robust recall responses did not impact antigen-specific NK cells, suggesting that adaptive and innate lymphocyte responses possess different activation requirements or occur in distinct anatomical locations. These findings have important implications in pathogen vaccination strategies that depend on the targeting of multiple T cell epitopes.

  12. Exquisite specificity and peptide epitope recognition promiscuity, properties shared by antibodies from sharks to humans.

    Science.gov (United States)

    Marchalonis, J J; Adelman, M K; Robey, I F; Schluter, S F; Edmundson, A B

    2001-01-01

    This review considers definitions of the specificity of antibodies including the development of recent concepts of recognition polyspecificity and epitope promiscuity. Using sets of homologous and unrelated peptides derived from the sequences of immunoglobulin and T cell receptor chains we offer operational definitions of cross-reactivity by investigating correlations of either identities in amino acid sequence, or in hydrophobicity/hydrophilicity profiles with degree of binding in enzyme-linked immunosorbent assays. Polyreactivity, or polyspecificity, are terms used to denote binding of a monoclonal antibody or purified antibody preparation to large complex molecules that are structurally unrelated, such as thyroglobulin and DNA. As a first approximation, there is a linear correlation between degree of sequence identity or hydrophobicity/hydrophilicity and antigenic cross-binding. However, catastrophic interchanges of amino acids can occur where changing of one amino acid out of 16 in a synthetic peptide essentially eliminates binding to certain antibodies. An operational definition of epitope promiscuity for peptides is the case where two peptides show little or no identity in amino acid sequence but bind strongly to the same antibody as shown by either direct binding or competitive inhibition. Analysis of antibodies of humans and sharks, the two most divergent species in evolution to express antibodies and the combinatorial immune response, indicates that the capacity for both exquisite specificity and epitope recognition promiscuity are essential conserved features of individual vertebrate antibodies.

  13. Prediction of epitopes using neural network based methods

    DEFF Research Database (Denmark)

    Lundegaard, Claus; Lund, Ole; Nielsen, Morten

    2011-01-01

    been evaluated to be among the very best performing MHC:peptide binding predictors available. Here we describe the background for these methods, and the rationale behind the different optimization steps implemented in the methods. We go through the practical use of the methods, which are publicly...... available in the form of relatively fast and simple web interfaces. Furthermore, we will review results obtained in actual epitope discovery projects where previous implementations of the described methods have been used in the initial selection of potential epitopes. Selected potential epitopes were all...

  14. Design and Characterization of Epitope-Scaffold Immunogens That Present the Motavizumab Epitope from Respiratory Syncytial Virus

    Energy Technology Data Exchange (ETDEWEB)

    McLellan, Jason S.; Correia, Bruno E.; Chen, Man; Yang, Yongping; Graham, Barney S.; Schief, William R.; Kwong, Peter D. (UWASH); (NIH)

    2012-06-28

    Respiratory syncytial virus (RSV) is a major cause of respiratory tract infections in infants, but an effective vaccine has not yet been developed. An ideal vaccine would elicit protective antibodies while avoiding virus-specific T-cell responses, which have been implicated in vaccine-enhanced disease with previous RSV vaccines. We propose that heterologous proteins designed to present RSV-neutralizing antibody epitopes and to elicit cognate antibodies have the potential to fulfill these vaccine requirements, as they can be fashioned to be free of viral T-cell epitopes. Here we present the design and characterization of three epitope-scaffolds that present the epitope of motavizumab, a potent neutralizing antibody that binds to a helix-loop-helix motif in the RSV fusion glycoprotein. Two of the epitope-scaffolds could be purified, and one epitope-scaffold based on a Staphylococcus aureus protein A domain bound motavizumab with kinetic and thermodynamic properties consistent with the free epitope-scaffold being stabilized in a conformation that closely resembled the motavizumab-bound state. This epitope-scaffold was well folded as assessed by circular dichroism and isothermal titration calorimetry, and its crystal structure (determined in complex with motavizumab to 1.9 {angstrom} resolution) was similar to the computationally designed model, with all hydrogen-bond interactions critical for binding to motavizumab preserved. Immunization of mice with this epitope-scaffold failed to elicit neutralizing antibodies but did elicit sera with F binding activity. The elicitation of F binding antibodies suggests that some of the design criteria for eliciting protective antibodies without virus-specific T-cell responses are being met, but additional optimization of these novel immunogens is required.

  15. Construction and expression of a chimeric gene with T-/B-cell epitopes of major allergen group 1 from Dermatophagoides farina%粉尘螨1类变应原T和B细胞表位嵌合基因的构建与表达

    Institute of Scientific and Technical Information of China (English)

    徐海丰; 徐朋飞; 王克霞; 李朝品

    2014-01-01

    目的:构建粉尘螨1类变应原(Der f 1)的T和B细胞表位嵌合基因原核表达载体。方法将Der f 1包含的5个T细胞表位(T1~T5)和6个B细胞表位(B1~B6),以B1-T1-B2-T2-B3-T3-B4-T4-B5-T5-B6和B1-B2-B3-B4-B5-B6-T1-T2-T3-T4-T5连接方式直接合成表位嵌合基因,并分别命名为Der f 1A和Der f 1B。构建原核表达重组质粒pET-28a(+)-Der f 1A和pET-28a(+)-Der f 1B,双酶切及测序验证的阳性克隆转化到E.coli BL21(DE3)后诱导表达。SDS-PAGE分析表达产物后进行纯化,并进行Western blotting鉴定。ELISA法检测嵌合蛋白对粉尘螨过敏患者血清IgE抗体的结合力。结果双酶切及测序结果表明,成功构建了原核表达重组质粒pET-28a(+)-Der f 1A和pET-28a(+)-Der f 1B;SDS-PAGE分析表明,Der f 1A和Der f 1B诱导并纯化成功,Western blotting结果进一步证实纯化了Der f 1A和Der f 1B嵌合蛋白。与Der f 1相比,Der f 1A和Der f 1B嵌合蛋白与粉尘螨过敏患者血清IgE抗体的结合能力显著降低(P均<0.05),但Der f 1A和Der f 1B间差异无统计学意义(P>0.05)。结论成功表达了Der f 1的T和B细胞表位嵌合蛋白,为粉尘螨过敏的特异性免疫治疗奠定了基础。%Objective To construct and express a chimeric gene with T-/B-cell epitopes of the major allergen group 1 from Dermatophagoides farina(Der f 1). Methods Two chimeric genes,Der f 1A and Der f 1B,were synthesized as B1-T1-B2-T2-B3-T3-B4-T4-B5-T5-B6 and B1-B2-B3-B4-B5-B6-T1-T2-T3-T4-T5 pattens. Two recombinant vectors,pET-28a(+)-Der f 1A and pET-28a(+)-Der f 1B,were constructed for prokaryotic expression. These chimeric genes were induced by 1 mmol/L IPTG (final concentration),digested with restriction enzymes and sequenced. The chimeric proteins were analyzed by SDS-PAGE and Western blotting. Results After digestion by restriction enzymes and sequencing,the recombinant vectors were constructed successfully. The

  16. Identification and enhancement of HLA-A2.1-restricted CTL epitopes in a new human cancer antigen-POTE.

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    Yi-Hsiang Huang

    Full Text Available Identification of CD8(+ T cell epitopes that can induce T cells to kill tumor cells is a fundamental step for development of a peptide cancer vaccine. POTE protein is a newly identified cancer antigen that was found to be expressed in a wide variety of human cancers, including prostate, colon, lung, breast, ovary and pancreas. Here, we determined HLA-A2.1-restricted cytotoxic T lymphocyte (CTL epitopes in the POTE protein, and also designed enhanced epitopes by amino acid (AA substitutions. Five 9-mer peptides were first selected and their binding affinity to HLA-A2 molecules was measured by the T2 binding assay. POTE 272-280 and POTE 323-331 showed the strongest HLA-A2 binding affinity. AA substituted peptides POTE 252-9V (with valine at position 9, POTE 553-1Y (with tyrosine at position 1 and POTE 323-3F (with phenylalanine at position 3 conferred higher affinity for HLA-A2, and induced CTL responses cross-reactive with wild type antigens. While POTE 252-9V was the strongest in this respect, POTE 323-3F had the greatest increase in immunogenicity compared to wild type. Importantly, two modified epitopes (POTE-553-1Y and POTE-323-3F induced CTLs that killed NCI-H522, a POTE-expressing HLA-A2(+ human non-small cell lung cancer cell line, indicating natural endogenous processing of these epitopes. In conclusion, the immunogenicity of POTE epitopes can be enhanced by peptide modification to induce T cells that kill human cancer cells. A combination of POTE 553-1Y and POTE 323-3F epitopes might be an attractive vaccine strategy for HLA-A2 cancer patients to overcome tolerance induced by tumors and prevent escape.

  17. An assessment on epitope prediction methods for protozoa genomes

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    Resende Daniela M

    2012-11-01

    Full Text Available Abstract Background Epitope prediction using computational methods represents one of the most promising approaches to vaccine development. Reduction of time, cost, and the availability of completely sequenced genomes are key points and highly motivating regarding the use of reverse vaccinology. Parasites of genus Leishmania are widely spread and they are the etiologic agents of leishmaniasis. Currently, there is no efficient vaccine against this pathogen and the drug treatment is highly toxic. The lack of sufficiently large datasets of experimentally validated parasites epitopes represents a serious limitation, especially for trypanomatids genomes. In this work we highlight the predictive performances of several algorithms that were evaluated through the development of a MySQL database built with the purpose of: a evaluating individual algorithms prediction performances and their combination for CD8+ T cell epitopes, B-cell epitopes and subcellular localization by means of AUC (Area Under Curve performance and a threshold dependent method that employs a confusion matrix; b integrating data from experimentally validated and in silico predicted epitopes; and c integrating the subcellular localization predictions and experimental data. NetCTL, NetMHC, BepiPred, BCPred12, and AAP12 algorithms were used for in silico epitope prediction and WoLF PSORT, Sigcleave and TargetP for in silico subcellular localization prediction against trypanosomatid genomes. Results A database-driven epitope prediction method was developed with built-in functions that were capable of: a removing experimental data redundancy; b parsing algorithms predictions and storage experimental validated and predict data; and c evaluating algorithm performances. Results show that a better performance is achieved when the combined prediction is considered. This is particularly true for B cell epitope predictors, where the combined prediction of AAP12 and BCPred12 reached an AUC value

  18. Identification of human leukemia antigen A*0201-restricted epitopes derived from epidermal growth factor pathway substrate number 8.

    Science.gov (United States)

    Tang, Baishan; Zhou, Weijun; Du, Jingwen; He, Yanjie; Li, Yuhua

    2015-08-01

    T-cell-mediated immunotherapy of hematological malignancies requires selection of targeted tumor-associated antigens and T-cell epitopes contained in these tumor proteins. Epidermal growth factor receptor pathway substrate 8 (EPS8), whose function is pivotal for tumor proliferation, progression and metastasis, has been found to be overexpressed in most human tumor types, while its expression in normal tissue is low. The aim of the present study was to identify human leukemia antigen (HLA)-A*0201-restricted epitopes of EPS8 by using a reverse immunology approach. To achieve this, computer algorithms were used to predict HLA-A*0201 molecular binding, proteasome cleavage patterns as well as translocation of transporters associated with antigen processing. Candidate peptides were experimentally validated by T2 binding affinity assay and brefeldin-A decay assay. The functional avidity of peptide-specific cytotoxic T lymphocytes (CTLs) induced from peripheral blood mononuclear cells of healthy volunteers were evaluated by using an enzyme-linked immunosorbent spot assay and a cytotoxicity assay. Four peptides, designated as P455, P92, P276 and P360, had high affinity and stability of binding towards the HLA-A*0201 molecule, and specific CTLs induced by them significantly responded to the corresponding peptides and secreted IFN-γ. At the same time, the CTLs were able to specifically lyse EPS8-expressing cell lines in an HLA-A*0201-restricted manner. The present study demonstrated that P455, P92, P276 and P360 were CTL epitopes of EPS8, and were able to be used for epitope-defined adoptive T-cell transfer and multi-epitope-based vaccine design.

  19. The Fas antigen (CD95) on human lymphoid cells: epitope analysis with ten antibodies.

    Science.gov (United States)

    Zola, H; Fusco, M; Ridings, J; Flego, L R; Weedon, H M; Nicholson, I; Organ, N; Roberton, D M; Macardle, P J

    1996-11-01

    The expression of CD95 antigen was examined on adult and cord blood lymphocytes using a highly sensitive immunofluorescence/flow cytometric procedure. CD95 was expressed by the majority of circulating blood T cells in adults, and by a smaller proportion of CD4+ and CD8+ T cells in cord blood. The majority of circulating B cells did not react with seven CD95 antibodies, but three antibodies did stain B cells. In tonsil sections, CD95 was expressed throughout the tissue but germinal centres showed generally stronger staining than the surrounding follicular mantle and interfollicular areas. This was confirmed by flow cytometry, which showed expression preferentially on B cells with a germinal centre phenotype. Because different antibodies stained different proportions of B cells, CD95 epitopes were examined by inhibition, additive binding and protease susceptibility studies using a panel of ten CD95 antibodies. B cells apparently reacting selectively with CD95 antibodies were sorted and CD95 mRNA was reverse transcribed to cDNA and analyzed, in order to confirm the presence of CD95 in cells which reacted selectively and to explore the possible existence of CD95 isoforms. The major cDNA band was identical in the two populations. Inhibition of N-glycosylation suggested that the epitopes detected differentially could not be accounted for by differential N-glycosylation.

  20. Strategic Use of Epitope Matching to Improve Outcomes.

    Science.gov (United States)

    Wiebe, Chris; Nickerson, Peter

    2016-10-01

    Understanding the events leading to allorecognition and the subsequent effector pathways engaged is key for the development of strategies to prolong graft survival. Optimizing patient outcomes will require 2 major advancements: (1) minimizing premature death with a functioning graft in the patients with stable graft function, and (2) maximizing graft survival by avoiding the aforementioned allorecognition. This necessitates personalized immunosuppression to avoid known metabolic side effects, risk for infection, and malignancy, while holding the alloimmune system in check. Since the beginning of transplant a key strategy to achieve this goal is to minimize HLA mismatching between donor and recipient. What has not evolved is any refinement in our evaluation of HLA relatedness between donor and recipient when HLA mismatch exists. Donor-recipient HLA mismatch at the amino acid level can now be determined. These mismatches serve as potential epitopes for de novo donor specific antibody development and correlate with late rejection and graft loss. It is in this context that HLA epitope analysis is considered as a strategy to permit safe immunosuppression minimization to improve patient outcomes through: (1) improved allocation schemes that favor donor-recipient pairs with a low HLA epitope mismatch load (especially at the class II loci) or avoiding specific epitope mismatches known to be highly immunogenic and (2) immunosuppressive minimization in patients with low epitope mismatch loads or without highly immunogenic epitope mismatches.

  1. Computational elucidation of potential antigenic CTL epitopes in Ebola virus.

    Science.gov (United States)

    Dikhit, Manas R; Kumar, Santosh; Vijaymahantesh; Sahoo, Bikash R; Mansuri, Rani; Amit, Ajay; Yousuf Ansari, Md; Sahoo, Ganesh C; Bimal, Sanjiva; Das, Pradeep

    2015-12-01

    Cell-mediated immunity is important for the control of Ebola virus infection. We hypothesized that those HLA A0201 and HLA B40 restricted epitopes derived from Ebola virus proteins, would mount a good antigenic response. Here we employed an immunoinformatics approach to identify specific 9mer amino acid which may be capable of inducing a robust cell-mediated immune response in humans. We identified a set of 28 epitopes that had no homologs in humans. Specifically, the epitopes derived from NP, RdRp, GP and VP40 share population coverage of 93.40%, 84.15%, 74.94% and 77.12%, respectively. Based on the other HLA binding specificity and population coverage, seven novel promiscuous epitopes were identified. These 7 promiscuous epitopes from NP, RdRp and GP were found to have world-wide population coverage of more than 95% indicating their potential significance as useful candidates for vaccine design. Epitope conservancy analysis also suggested that most of the peptides are highly conserved (100%) in other virulent Ebola strain (Mayinga-76, Kikwit-95 and Makona-G3816- 2014) and can therefore be further investigated for their immunological relevance and usefulness as vaccine candidates.

  2. Development of a sensitive and specific epitope-blocking ELISA for universal detection of antibodies to human enterovirus 71 strains.

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    Fang He

    Full Text Available BACKGROUND: Human Enterovirus 71 (EV71 is a common cause of hand, foot and mouth disease (HFMD in young children. It is often associated with severe neurological diseases and mortalities in recent outbreaks across the Asia Pacific region. Currently, there is no efficient universal antibody test available to detect EV71 infections. METHODOLOGY/PRINCIPAL FINDING: In the present study, an epitope-blocking ELISA was developed to detect specific antibodies to human EV71 viruses in human or animal sera. The assay relies on a novel monoclonal antibody (Mab 1C6 that specifically binds to capsid proteins in whole EV71 viruses without any cross reaction to any EV71 capsid protein expressed alone. The sensitivity and specificity of the epitope-blocking ELISA for EV71 was evaluated and compared to microneutralization using immunized animal sera to multiple virus genotypes of EV71 and coxsackieviruses. Further, 200 serum sample from human individuals who were potentially infected with EV71 viruses were tested in both the blocking ELISA and microneutralization. Results indicated that antibodies to EV71 were readily detected in immunized animals or human sera by the epitope blocking ELISA whereas specimens with antibodies to other enteroviruses yielded negative results. This assay is not only simpler to perform but also shows higher sensitivity and specificity as compared to microneutralization. CONCLUSION: The epitope-blocking ELISA based on a unique Mab 1C6 provided highly sensitive and 100% specific detection of antibodies to human EV71 viruses in human sera.

  3. Epitope Mapping of Avian Influenza M2e Protein: Different Species Recognise Various Epitopes.

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    Noor Haliza Hasan

    Full Text Available A common approach for developing diagnostic tests for influenza virus detection is the use of mouse or rabbit monoclonal and/or polyclonal antibodies against a target antigen of the virus. However, comparative mapping of the target antigen using antibodies from different animal sources has not been evaluated before. This is important because identification of antigenic determinants of the target antigen in different species plays a central role to ensure the efficiency of a diagnostic test, such as competitive ELISA or immunohistochemistry-based tests. Interest in the matrix 2 ectodomain (M2e protein of avian influenza virus (AIV as a candidate for a universal vaccine and also as a marker for detection of virus infection in vaccinated animals (DIVA is the rationale for the selection of this protein for comparative mapping evaluation. This study aimed to map the epitopes of the M2e protein of avian influenza virus H5N1 using chicken, mouse and rabbit monoclonal or monospecific antibodies. Our findings revealed that rabbit antibodies (rAbs recognized epitope 6EVETPTRN13 of the M2e, located at the N-terminal of the protein, while mouse (mAb and chicken antibodies (cAbs recognized epitope 10PTRNEWECK18, located at the centre region of the protein. The findings highlighted the difference between the M2e antigenic determinants recognized by different species that emphasized the importance of comparative mapping of antibody reactivity from different animals to the same antigen, especially in the case of multi-host infectious agents such as influenza. The findings are of importance for antigenic mapping, as well as diagnostic test and vaccine development.

  4. Design and evaluation of a multi-epitope assembly Peptide (MEAP against herpes simplex virus type 2 infection in BALB/c mice

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    Pan Mingjie

    2011-05-01

    Full Text Available Abstract Background Human herpes simplex virus (HSV 1 and 2 causes oral, ocular, or genital infections, which remains a significant health problem worldwide. HSV-1 and -2 infections in humans range from localized skin infections of the oral, ocular, and genital regions to severe and often disseminated infections in immunocompromised hosts. Epitope based vaccination is a promising mean to achieve protective immunity and to avoid infections with Human herpes simplex virus type 2 (HSV-2. Methods The twelve selected epitopes, six B cell epitopes from different glycoprotein of HSV-2 (amino acid residues 466-473 (EQDRKPRN from envelope glycoprotein B, 216-223 (GRTDRPSA from C, 6-18 (DPSLKMADPNRFR from D, 483-491 (DPPERPDSP from E, 572-579 (EPPDDDDS from G and 286-295 (CRRRYRRPRG from I glycoprotein of HSV-2, four CD4+ T cell epitopes (amino acid residues 21-28 (NLPVLDQL from D, 162-177 (KDVTVSQVWFGHRYSQ from B, 205-224 (KAYQQGVTVDSIGMLPRFIP from D and 245-259 (KPPYTSTLLPPELSD from D and two CD8+ T cell epitopes (amino acid residues 10-20 (KMADPNRFRGK from D and 268-276 (ALLEDPAGT from D, are responsible for the elicitation of the neutralizing antibodies and cytotoxic T lymphocytes (CTLs that impart protective immunity to the host. In this study, all above epitopes were inserted into the extracellular fragment (amino acid residues 1-290 of HSV-2 glycoprotein D to construct multi-epitope assembly peptides (MEAPs by replacing some non-epitope amino acid sequences. The epitope independency of the MEAPs was predicted by three-dimensional software algorithms. The gene of the selected MEAP was expressed in E.coli BL21(DE3, and its protective efficacy against HSV-2 infection was assessed in BALB/c mice. Results The MEAP, with each inserted epitopes independently displayed on the molecule surface, was selected as candidate proteins. The results showed that the MEAP was highly immunogenic and could elicit high titer neutralizing antibodies and cell

  5. T cell epitope-based allergy vaccines.

    Science.gov (United States)

    Larché, Mark

    2011-01-01

    Specific immunotherapy (SIT) with extracts containing intact allergen molecules is clinically efficacious, but associated with frequent adverse events related to the allergic sensitization of the patient. As a result, treatment is initiated in an incremental dose fashion which ultimately achieves a plateau (maintenance dose) that may be continued for several years. Reduction of allergic adverse events may allow safer and more rapid treatment Thus, many groups have developed and evaluated strategies to reduce allergenicity whilst maintaining immunogenicity, the latter being required to achieve specific modulation of the immune response. Peptide immunotherapy can be used to target T and/or B cells in an antigen-specific manner. To date, only approaches that target T cells have been clinically evaluated. Short, synthetic peptides representing immunodominant T cell epitopes of major allergens are able to modulate allergen-specific T cell responses in the absence of IgE cross linking and activation of effector cells. Here we review clinical and mechanistic studies associated with peptide immunotherapy targeting allergy to cats or to bee venom. 

  6. Immunization with a peptide containing MHC class I and II epitopes derived from the tumor antigen SIM2 induces an effective CD4 and CD8 T-cell response.

    Directory of Open Access Journals (Sweden)

    Haydn T Kissick

    Full Text Available Here, we sought to determine whether peptide vaccines designed harbor both class I as well as class II restricted antigenic motifs could concurrently induce CD4 and CD8 T cell activation against autologous tumor antigens. Based on our prior genome-wide interrogation of human prostate cancer tissues to identify genes over-expressed in cancer and absent in the periphery, we targeted SIM2 as a prototype autologous tumor antigen for these studies. Using humanized transgenic mice we found that the 9aa HLA-A*0201 epitope, SIM2(237-245, was effective at inducing an antigen specific response against SIM2-expressing prostate cancer cell line, PC3. Immunization with a multi-epitope peptide harboring both MHC-I and MHC-II restricted epitopes induced an IFN-γ response in CD8 T cells to the HLA-A*0201-restricted SIM2(237-245 epitope, and an IL-2 response by CD4 T cells to the SIM2(240-254 epitope. This peptide was also effective at inducing CD8+ T-cells that responded specifically to SIM2-expressing tumor cells. Collectively, the data presented in this study suggest that a single peptide containing multiple SIM2 epitopes can be used to induce both a CD4 and CD8 T cell response, providing a peptide-based vaccine formulation for potential use in immunotherapy of various cancers.

  7. Steric shielding of surface epitopes and impaired immune recognition induced by the ebola virus glycoprotein.

    Directory of Open Access Journals (Sweden)

    Joseph R Francica

    Full Text Available Many viruses alter expression of proteins on the surface of infected cells including molecules important for immune recognition, such as the major histocompatibility complex (MHC class I and II molecules. Virus-induced downregulation of surface proteins has been observed to occur by a variety of mechanisms including impaired transcription, blocks to synthesis, and increased turnover. Viral infection or transient expression of the Ebola virus (EBOV glycoprotein (GP was previously shown to result in loss of staining of various host cell surface proteins including MHC1 and β1 integrin; however, the mechanism responsible for this effect has not been delineated. In the present study we demonstrate that EBOV GP does not decrease surface levels of β1 integrin or MHC1, but rather impedes recognition by steric occlusion of these proteins on the cell surface. Furthermore, steric occlusion also occurs for epitopes on the EBOV glycoprotein itself. The occluded epitopes in host proteins and EBOV GP can be revealed by removal of the surface subunit of GP or by removal of surface N- and O- linked glycans, resulting in increased surface staining by flow cytometry. Importantly, expression of EBOV GP impairs CD8 T-cell recognition of MHC1 on antigen presenting cells. Glycan-mediated steric shielding of host cell surface proteins by EBOV GP represents a novel mechanism for a virus to affect host cell function, thereby escaping immune detection.

  8. An immunoinformatic approach for identification of Trypanosoma cruzi HLA-A2-restricted CD8+ T cell epitopes

    Science.gov (United States)

    Eickhoff, Christopher S; Van Aartsen, Daniel; Terry, Frances E; Meymandi, Sheba K; Traina, Mahmoud M; Hernandez, Salvador; Martin, William D; Moise, Leonard; De Groot, Annie S; Hoft, Daniel F

    2015-01-01

    Chagas disease is a major neglected tropical disease caused by persistent chronic infection with the protozoan parasite Trypanosoma cruzi. An estimated 8 million people are infected with T. cruzi, however only 2 drugs are approved for treatment and no vaccines are available. Thus there is an urgent need to develop vaccines and new drugs to prevent and treat Chagas disease. In this work, we identify T cell targets relevant for human infection with T. cruzi. The trans-sialidase (TS) gene family is a large family of homologous genes within the T. cruzi genome encoding over 1,400 members. There are 12 highly conserved TS gene family members which encode enzymatically active TS (functional TS; F-TS), while the remaining TS family genes are less conserved, enzymatically inactive and have been hypothesized to be involved in immune evasion (non-functional TS; NF-TS). We utilized immunoinformatic tools to identify HLA-A2-restricted CD8+ T cell epitopes conserved within F-TS family members and NF-TS gene family members. We also utilized a whole-genome approach to identify T cell epitopes present within genes which have previously been shown to be expressed in life stages relevant for human infection (Non-TS genes). Thirty immunogenic HLA-A2-restricted CD8+ T cell epitopes were identified using IFN-γ ELISPOT assays after vaccination of humanized HLA-A2 transgenic mice with mature dendritic cells pulsed with F-TS, NF-TS, and Non-TS peptide pools. The immunogenic HLA-A2-restricted T cell epitopes identified in this work may serve as potential components of an epitope-based T cell targeted vaccine for Chagas disease. PMID:26107442

  9. Localization of key amino acid residues in the dominant conformational epitopes on thyroid peroxidase recognized by mouse monoclonal antibodies.

    Science.gov (United States)

    Godlewska, Marlena; Czarnocka, Barbara; Gora, Monika

    2012-09-01

    Autoantibodies to thyroid peroxidase (TPO), the major target autoantigen in autoimmune thyroid diseases, recognize conformational epitopes limited to two immunodominant regions (IDRs) termed IDR-A and -B. The apparent restricted heterogeneity of TPO autoantibodies was discovered using TPO-specific mouse monoclonal antibodies (mAbs) and later confirmed by human recombinant Fabs. In earlier studies we identified key amino acids crucial for the interaction of human autoantibodies with TPO. Here we show the critical residues that participate in binding of five mAbs to the conformational epitopes on the TPO surface. Using ELISA we tested the reactivity of single and multiple TPO mutants expressed in CHO cells with a panel of mAbs specifically recognizing IDR-A (mAb 2 and 9) and IDR-B (mAb 15, 18, 64). We show that antibodies recognizing very similar regions on the TPO surface may interact with different sets of residues. We found that residues K713 and E716 contribute to the interaction between mAb 2 and TPO. The epitope for mAb 9 is critically dependent on residues R646 and E716. Moreover, we demonstrate that amino acids E604 and D630 are part of the functional epitope for mAb 15, and amino acids D624 and K627 for mAb 18. Finally, residues E604, D620, D624, K627, and D630 constitute the epitope for mAb 64. This is the first detailed study identifying the key resides for binding of mAbs 2, 9, 15, 18, and 64. Better understanding of those antibodies' specificity will be helpful in elucidating the properties of TPO as an antigen in autoimmune disorders.

  10. Molecular Characterization of Two Monoclonal Antibodies against the Same Epitope on B-Cell Receptor Associated Protein 31.

    Science.gov (United States)

    Kim, Won-Tae; Shin, Saemina; Hwang, Hyo Jeong; Kim, Min Kyu; Jung, Han-Sung; Park, Hwangseo; Ryu, Chun Jeih

    2016-01-01

    Previously, we showed that B-cell receptor associated protein 31 (BAP31), an endoplasmic reticulum (ER) membrane chaperone, is also expressed on the cell surface by two monoclonal antibodies (MAbs) 297-D4 and 144-A8. Both MAbs recognize the same linear epitope on the C-terminal domain of BAP31, although they were independently established. Here, flow cytometric analysis showed that 144-A8 had additional binding properties to some cells, as compared to 297-D4. Quantitative antigen binding assays also showed that 144-A8 had higher antigen binding capacity than 297-D4. Affinity measurement revealed that 144-A8 had 1.54-fold higher binding affinity than 297-D4. Analysis of the heavy- and light-chain variable region sequences of two MAbs revealed that both MAbs belonged to the same heavy chain (Igh-V3660 VH3) and light chain subgroup (IGKV21) with just two amino acid differences in each framework region, indicating that both MAbs arise from the same germline origin. Seven amino acid differences were found between the complementarity determining regions (CDRs) of the two MAbs. Molecular modeling of the epitope-paratope complexes revealed that the epitope appeared to reside in closer proximity to the CDRs of 144-A8 than to those of 297-D4 with the stronger hydrogen bond interactions with the former than the latter. More interestingly, an additional hydrophobic interaction appeared to be established between the leucine residue of epitope and the paratope of 144-A8, due to the substitution of H-Tyr101 for H-Phe101 in 144-A8. Thus, the different binding specificity and affinity of 144-A8 appeared to be due to the different hydrogen bonds and hydrophobic interaction induced by the alterations of amino acids in CDRs of 144-A8. The results provide molecular insights into how the binding specificities and affinities of antibodies evolve with the same epitope in different microenvironments.

  11. IL-7 Induces an Epitope Masking of γc Protein in IL-7 Receptor Signaling Complex

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    Tae Sik Goh

    2017-01-01

    Full Text Available IL-7 signaling via IL-7Rα and common γ-chain (γc is necessary for the development and homeostasis of T cells. Although the delicate mechanism in which IL-7Rα downregulation allows the homeostasis of T cell with limited IL-7 has been well known, the exact mechanism behind the interaction between IL-7Rα and γc in the absence or presence of IL-7 remains unclear. Additionally, we are still uncertain as to how only IL-7Rα is separately downregulated by the binding of IL-7 from the IL-7Rα/γc complex. We demonstrate here that 4G3, TUGm2, and 3E12 epitope masking of γc protein are induced in the presence of IL-7, indicating that the epitope alteration is induced by IL-7 binding to the preassembled receptor core. Moreover, the epitope masking of γc protein is inversely correlated with the expression of IL-7Rα upon IL-7 binding, implying that the structural alteration of γc might be involved in the regulation of IL-7Rα expression. The conformational change in γc upon IL-7 binding may contribute not only to forming the functional IL-7 signaling complex but also to optimally regulating the expression of IL-7Rα.

  12. High Throughput T Epitope Mapping and Vaccine Development

    Directory of Open Access Journals (Sweden)

    Giuseppina Li Pira

    2010-01-01

    Full Text Available Mapping of antigenic peptide sequences from proteins of relevant pathogens recognized by T helper (Th and by cytolytic T lymphocytes (CTL is crucial for vaccine development. In fact, mapping of T-cell epitopes provides useful information for the design of peptide-based vaccines and of peptide libraries to monitor specific cellular immunity in protected individuals, patients and vaccinees. Nevertheless, epitope mapping is a challenging task. In fact, large panels of overlapping peptides need to be tested with lymphocytes to identify the sequences that induce a T-cell response. Since numerous peptide panels from antigenic proteins are to be screened, lymphocytes available from human subjects are a limiting factor. To overcome this limitation, high throughput (HTP approaches based on miniaturization and automation of T-cell assays are needed. Here we consider the most recent applications of the HTP approach to T epitope mapping. The alternative or complementary use of in silico prediction and experimental epitope definition is discussed in the context of the recent literature. The currently used methods are described with special reference to the possibility of applying the HTP concept to make epitope mapping an easier procedure in terms of time, workload, reagents, cells and overall cost.

  13. Identification of a Novel Immunodominant HLA-B*07: 02-restricted Adenoviral Peptide Epitope and Its Potential in Adoptive Transfer Immunotherapy.

    Science.gov (United States)

    Günther, Patrick S; Peper, Janet K; Faist, Benjamin; Kayser, Simone; Hartl, Lena; Feuchtinger, Tobias; Jahn, Gerhard; Neuenhahn, Michael; Busch, Dirk H; Stevanović, Stefan; Dennehy, Kevin M

    2015-09-01

    Adenovirus infections of immunocompromised patients, particularly following allogeneic hematopoietic stem cell transplantation, are associated with morbidity and mortality. Immunotherapy by adoptive transfer of hexon-specific and penton-specific T cells has been successfully applied, but many approaches are impeded by the low number of HLA class I-restricted adenoviral peptide epitopes described to date. We use a novel method to identify naturally presented adenoviral peptide epitopes from infected human cells, ectopically expressing defined HLA, using peptide elution and liquid chromatography-mass spectrometry analysis. We show that the previously described HLA-A*01:01-restricted peptide epitope LTDLGQNLLY from hexon protein is naturally presented, and demonstrate the functionality of LTDLGQNLLY-specific T cells. We further identify a novel immunodominant HLA-B*07:02-restricted peptide epitope VPATGRTLVL from protein 13.6 K, and demonstrate the high proliferative, cytotoxic, and IFN-γ-producing capacity of peptide-specific T cells. Lastly, LTDLGQNLLY-specific T cells can be detected ex vivo following adoptive transfer therapy, and LTDLGQNLLY-specific and VPATGRTLVL-specific T cells have memory phenotypes ex vivo. Given their proliferative and cytotoxic capacity, such epitope-specific T cells are promising candidates for adoptive T-cell transfer therapy of adenovirus infection.

  14. Dengue virus-reactive CD8+ T cells display quantitative and qualitative differences in their response to variant epitopes of heterologous viral serotypes.

    Science.gov (United States)

    Bashyam, Hema S; Green, Sharone; Rothman, Alan L

    2006-03-01

    Reactivation of serotype cross-reactive CD8+ memory T lymphocytes is thought to contribute to the immunopathogenesis of dengue disease during secondary infection by a heterologous serotype. Using cytokine flow cytometry, we have defined four novel HLA-A*02-restricted dengue viral epitopes recognized by up to 1.5% of circulating CD8+ T cells in four donors after primary vaccination. All four donors had the highest cytokine response to the epitope NS4b 2353. We also studied the effect of sequence differences in heterologous dengue serotypes on dengue-reactive CD8+ memory T cell cytokine and proliferative responses. The D3 variant of a different NS4b epitope 2423 and the D2 variant of the NS4a epitope 2148 induced the largest cytokine response, compared with their respective heterologous sequences in all donors regardless of the primary vaccination serotype. Stimulation with variant peptides also altered the relative frequencies of the various subsets of cells that expressed IFN-gamma, TNF-alpha, MIP-1beta, and combinations of these cytokines. These results indicate that the prior infection history of the individual as well as the serotypes of the primary and heterologous secondary viruses influence the nature of the secondary response. These differences in the effector functions of serotype cross-reactive memory T cells induced by heterologous variant epitopes, which are both quantitative and qualitative, may contribute to the clinical outcome of secondary dengue infection.

  15. Epitope mapping on the dendritic cell-specific ICAM-3-grabbing non-integrin (DC-SIGN) pathogen-attachment factor.

    Science.gov (United States)

    Sierra-Filardi, Elena; Estecha, Ana; Samaniego, Rafael; Fernández-Ruiz, Elena; Colmenares, María; Sánchez-Mateos, Paloma; Steinman, Ralph M; Granelli-Piperno, Angela; Corbí, Angel L

    2010-01-01

    DC-SIGN (dendritic cell-specific ICAM-3-grabbing non-integrin) is a myeloid pathogen-attachment factor C-type lectin which recognizes mannose- and fucose-containing oligosaccharide ligands on clinically relevant pathogens. Intracellular signaling initiated upon ligand engagement of DC-SIGN interferes with TLR-initiated signals, and modulates the T cell activating and polarizing ability of antigen-presenting cells. The C-terminal carbohydrate-recognition domain (CRD) of DC-SIGN is preceded by a neck domain composed of eight 23-residue repeats which mediate molecule multimerization, and whose polymorphism correlates with altered susceptibility to SARS and HIV infection. Naturally occurring isoforms and chimaeric molecules, in combination with established recognition properties, were used to define seven structural and functional epitopes on DC-SIGN. Three epitopes mapped to the CRD, one of which is multimerization-dependent and only exposed on DC-SIGN monomers. Epitopes within the neck domain were conformation-independent and unaltered upon molecule multimerization, but were differentially affected by neck domain truncations. Although neck-specific antibodies exhibited lower function-blocking ability, they were more efficient at inducing molecule internalization. Moreover, crosslinking of the different epitopes resulted in distinct levels of microclustering on the cell surface. The identification of independent epitopes on the DC-SIGN molecule might facilitate the design of reagents that modulate the T cell activating and polarizing ability of DC-SIGN-expressing cells without preventing its antigen- and pathogen-recognition capacities.

  16. Phage displaying epitope of Candida albicans HSP90 and serodiagnosis

    Institute of Scientific and Technical Information of China (English)

    杨琼; 王丽; 卢大宁; 邢沈阳; 尹东; 朱筱娟

    2004-01-01

    @@ Recently, the frequent use of immunosuppressants and chemotherapeutic drugs for cancers has caused an increase in the frequency of life-threatening systemic candidiasis.1 Studies by Matthews et al2 indicated HSP90 fragments are major targets for the immune system in infection due to C. albicans, and anti-epitope LKVIRK of HSP90 antibody is a serological marker for diagnosis of invasive candidiasis. Cloning and sequencing HSP90 antigen revealed that the linear epitope LKVIRK, localized near the C-terminus of the 47 kDa protein which circulates in the sera of patients with invasive candidiasis, as a heat-stable breakdown product of large more heat-labile antigen HSP90.2 In this study, epitope LKVIRK was displayed on the surface of phage fd to develop a new serological test for systemic candidiasis.

  17. Antibody specific epitope prediction-emergence of a new paradigm.

    Science.gov (United States)

    Sela-Culang, Inbal; Ofran, Yanay; Peters, Bjoern

    2015-04-01

    The development of accurate tools for predicting B-cell epitopes is important but difficult. Traditional methods have examined which regions in an antigen are likely binding sites of an antibody. However, it is becoming increasingly clear that most antigen surface residues will be able to bind one or more of the myriad of possible antibodies. In recent years, new approaches have emerged for predicting an epitope for a specific antibody, utilizing information encoded in antibody sequence or structure. Applying such antibody-specific predictions to groups of antibodies in combination with easily obtainable experimental data improves the performance of epitope predictions. We expect that further advances of such tools will be possible with the integration of immunoglobulin repertoire sequencing data.

  18. Common food allergens and their IgE-binding epitopes.

    Science.gov (United States)

    Matsuo, Hiroaki; Yokooji, Tomoharu; Taogoshi, Takanori

    2015-10-01

    Food allergy is an adverse immune response to certain kinds of food. Although any food can cause allergic reactions, chicken egg, cow's milk, wheat, shellfish, fruit, and buckwheat account for 75% of food allergies in Japan. Allergen-specific immunoglobulin E (IgE) antibodies play a pivotal role in the development of food allergy. Recent advances in molecular biological techniques have enabled the efficient analysis of food allergens. As a result, many food allergens have been identified, and their molecular structure and IgE-binding epitopes have also been identified. Studies of allergens have demonstrated that IgE antibodies specific to allergen components and/or the peptide epitopes are good indicators for the identification of patients with food allergy, prediction of clinical severity and development of tolerance. In this review, we summarize our current knowledge regarding the allergens and IgE epitopes in the well-researched allergies to chicken egg, cow's milk, wheat, shrimp, and peanut.

  19. Mature Epitope Density - A strategy for target selection based on immunoinformatics and exported prokaryotic proteins

    DEFF Research Database (Denmark)

    Santos, Anderson R; Pereira, Vanessa Bastos; Barbosa, Eudes;

    2013-01-01

    BACKGROUND: Current immunological bioinformatic approaches focus on the prediction of allele-specific epitopes capable of triggering immunogenic activity. The prediction of major histocompatibility complex (MHC) class I epitopes is well studied, and various software solutions exist for this purpo...

  20. Branched peptide amphiphiles, related epitope compounds and self assembled structures thereof

    Science.gov (United States)

    Stupp, Samuel I.; Guler, Mustafa O.

    2008-11-18

    Branched peptide amphiphilic compounds incorporating one or residues providing a pendant amino group for coupling one or more epitope sequences thereto, such compounds and related compositions for enhanced epitope presentation.

  1. Adenovirus-based vaccine with epitopes incorporated in novel fiber sites to induce protective immunity against Pseudomonas aeruginosa.

    Science.gov (United States)

    Sharma, Anurag; Krause, Anja; Xu, Yaqin; Sung, Biin; Wu, Wendy; Worgall, Stefan

    2013-01-01

    Adenovirus (Ad) vector-based vaccines displaying pathogen-derived epitopes on Ad capsid proteins can elicit anti-pathogen immunity. This approach seems to be particularly efficient with epitopes incorporated into the Ad fiber protein. Here, we explore epitope insertion into various sites of the Ad fiber to elicit epitope-specific immunity. Ad vectors expressing the 14-mer Pseudomonas aeruginosa immune-dominant outer membrane protein F (OprF) epitope 8 (Epi8) in five distinct sites of the Ad5 fiber, loops CD (AdZ.F(CD)Epi8), DE (AdZ.F(DE)Epi8), FG (AdZ.F(FG)Epi8), HI (AdZ.F(HI)Epi8) and C terminus (AdZ.F(CT)Epi8), or the hexon HVR5 loop (AdZ.HxEpi8) were compared in their capacity to elicit anti-P. aeruginosa immunity to AdOprF, an Ad expressing the entire OprF protein. Intramuscular immunization of BALB/c mice with AdZ.F(FG)Epi8 or AdZ.F(HI)Epi8 elicited higher anti-OprF humoral and cellular CD4 and CD8 responses as well as enhanced protection against respiratory infection with P. aeruginosa compared to immunization with AdZ.F(CD)Epi8, AdZ.F(DE)Epi8, AdZ.F(CT)Epi8 or AdZ.HxEpi8. Importantly, repeat administration of the fiber- and hexon-modified Ad vectors boosted the OprF-specific humoral immune response in contrast to immunization with AdOprF. Strikingly, following three doses of AdZ.F(FG)Epi8 or AdZ.F(HI)Epi8 anti-OprF immunity surpassed that induced by AdOprF. Furthermore, in the presence of anti-Ad5 immunity, immunization with AdZ.F(FG)Epi8 or AdZ.F(HI)Epi8, but not with AdOprF, induced protective immunity against P. aeruginosa. This suggests that incorporation of epitopes into distinct sites of the Ad fiber is a promising vaccine strategy.

  2. Antibody protection reveals extended epitopes on the human TSH receptor.

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    Rauf Latif

    Full Text Available Stimulating, and some blocking, antibodies to the TSH receptor (TSHR have conformation-dependent epitopes reported to involve primarily the leucine rich repeat region of the ectodomain (LRD. However, successful crystallization of TSHR residues 22-260 has omitted important extracellular non-LRD residues including the hinge region which connects the TSHR ectodomain to the transmembrane domain and which is involved in ligand induced signal transduction. The aim of the present study, therefore, was to determine if TSHR antibodies (TSHR-Abs have non-LRD binding sites outside the LRD. To obtain this information we employed the method of epitope protection in which we first protected TSHR residues 1-412 with intact TSHR antibodies and then enzymatically digested the unprotected residues. Those peptides remaining were subsequently delineated by mass spectrometry. Fourteen out of 23 of the reported stimulating monoclonal TSHR-Ab crystal contact residues were protected by this technique which may reflect the higher binding energies of certain residues detected in this approach. Comparing the protected epitopes of two stimulating TSHR-Abs we found both similarities and differences but both antibodies also contacted the hinge region and the amino terminus of the TSHR following the signal peptide and encompassing cysteine box 1 which has previously been shown to be important for TSH binding and activation. A monoclonal blocking TSHR antibody revealed a similar pattern of binding regions but the residues that it contacted on the LRD were again distinct. These data demonstrated that conformationally dependent TSHR-Abs had epitopes not confined to the LRDs but also incorporated epitopes not revealed in the available crystal structure. Furthermore, the data also indicated that in addition to overlapping contact regions within the LRD, there are unique epitope patterns for each of the antibodies which may contribute to their functional heterogeneity.

  3. Identification of two linear B-cell epitopes from West Nile virus NS1 by screening a phage-displayed random peptide library

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    Qin Yong-Li

    2011-07-01

    Full Text Available Abstract Background The West Nile virus (WNV nonstructural protein 1 (NS1 is an important antigenic protein that elicits protective antibody responses in animals and can be used for the serological diagnosis of WNV infection. Although previous work has demonstrated the vital role of WNV NS1-specific antibody responses, the specific epitopes in the NS1 have not been identified. Results The present study describes the identification of two linear B-cell epitopes in WNV NS1 through screening a phage-displayed random 12-mer peptide library with two monoclonal antibodies (mAbs 3C7 and 4D1 that directed against the NS1. The mAbs 3C7 and 4D1 recognized phages displaying peptides with the consensus motifs LTATTEK and VVDGPETKEC, respectively. Exact sequences of both motifs were found in the NS1 (895LTATTEK901 and 925VVDGPETKEC934. Further identification of the displayed B cell epitopes were conducted using a set of truncated peptides expressed as MBP fusion proteins. The data indicated that 896TATTEK901 and925VVDGPETKEC934 are minimal determinants of the linear B cell epitopes recognized by the mAbs 3C7 and 4D1, respectively. Antibodies present in the serum of WNV-positive horses recognized the minimal linear epitopes in Western blot analysis, indicating that the two peptides are antigenic in horses during infection. Furthermore, we found that the epitope recognized by 3C7 is conserved only among WNV strains, whereas the epitope recognized by 4D1 is a common motif shared among WNV and other members of Japanese encephalitis virus (JEV serocomplex. Conclusions We identified TATTEK and VVDGPETKEC as NS1-specific linear B-cell epitopes recognized by the mAbs 3C7 and 4D1, respectively. The knowledge and reagents generated in this study may have potential applications in differential diagnosis and the development of epitope-based marker vaccines against WNV and other viruses of JEV serocomplex.

  4. Oral immunotherapy for pollen allergy using T-cell epitope-containing egg white derived from genetically manipulated chickens.

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    Yoshinori Kawabe

    Full Text Available Peptide immunotherapy using T-cell epitopes is expected to be an effective treatment for allergic diseases such as Japanese cedar (Cryptomeria japonica; Cj pollinosis. To develop a treatment for pollen allergy by inducing oral tolerance, we generated genetically manipulated (GM chickens by retroviral gene transduction, to produce a fusion protein of chicken egg white lysozyme and a peptide derived from seven dominant human T-cell epitopes of Japanese cedar pollen allergens (cLys-7crp. The transgene sequence was detected in all chickens transduced with the retroviral vector. Transduction efficiency in blood cells correlated to transgene expression. Western blot analysis revealed that cLys-7crp was expressed in the egg white of GM hens. Mice induced to develop allergic rhinitis by Cj pollinosis were fed with cLys-7crp-containing egg white produced by GM chickens. Total and Cj allergen (Cry j 1-specific IgE levels were significantly decreased in allergic mice fed with cLys-7crp-containing egg white compared with allergic mice fed with normal egg white. These results suggest that oral administration of T-cell epitope-containing egg white derived from GM chickens is effective for the induction of immune tolerance as an allergy therapy.

  5. High-throughput epitope identification for snakebite antivenom

    DEFF Research Database (Denmark)

    Engmark, Mikael; De Masi, Federico; Laustsen, Andreas Hougaard

    Insight into the epitopic recognition pattern for polyclonal antivenoms is a strong tool for accurate prediction of antivenom cross-reactivity and provides a basis for design of novel antivenoms. In this work, a high-throughput approach was applied to characterize linear epitopes in 966 individua...... toxins from pit vipers (Crotalidae) using the ICP Crotalidae antivenom. Due to an abundance of snake venom metalloproteinases and phospholipase A2s in the venoms used for production of the investigated antivenom, this study focuses on these toxin families....

  6. Epitope mapping for monoclonal antibody reveals the activation mechanism for αVβ3 integrin.

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    Tetsuji Kamata

    Full Text Available Epitopes for a panel of anti-αVβ3 monoclonal antibodies (mAbs were investigated to explore the activation mechanism of αVβ3 integrin. Experiments utilizing αV/αIIb domain-swapping chimeras revealed that among the nine mAbs tested, five recognized the ligand-binding β-propeller domain and four recognized the thigh domain, which is the upper leg of the αV chain. Interestingly, the four mAbs included function-blocking as well as non-functional mAbs, although they bound at a distance from the ligand-binding site. The epitopes for these four mAbs were further determined using human-to-mouse αV chimeras. Among the four, P3G8 recognized an amino acid residue, Ser-528, located on the side of the thigh domain, while AMF-7, M9, and P2W7 all recognized a common epitope, Ser-462, that was located close to the α-genu, where integrin makes a sharp bend in the crystal structure. Fibrinogen binding studies for cells expressing wild-type αVβ3 confirmed that AMF-7, M9, and P2W7 were inhibitory, while P3G8 was non-functional. However, these mAbs were all unable to block binding when αVβ3 was constrained in its extended conformation. These results suggest that AMF-7, M9, and P2W7 block ligand binding allosterically by stabilizing the angle of the bend in the bent conformation. Thus, a switchblade-like movement of the integrin leg is indispensable for the affinity regulation of αVβ3 integrin.

  7. Human immune responses to H. pylori HLA Class II epitopes identified by immunoinformatic methods.

    Directory of Open Access Journals (Sweden)

    Songhua Zhang

    Full Text Available H. pylori persists in the human stomach over decades and promotes several adverse clinical sequelae including gastritis, peptic ulcers and gastric cancer that are linked to the induction and subsequent evasion of chronic gastric inflammation. Emerging evidence indicates that H. pylori infection may also protect against asthma and some other immune-mediated conditions through regulatory T cell effects outside the stomach. To characterize the complexity of the CD4+ T cell response generated during H. pylori infection, computational methods were previously used to generate a panel of 90 predicted epitopes conserved among H. pylori genomes that broadly cover HLA Class II diversity for maximum population coverage. Here, these sequences were tested individually for their ability to induce in vitro responses in peripheral blood mononuclear cells by interferon-γ ELISpot assay. The average number of spot-forming cells/million PBMCs was significantly elevated in H. pylori-infected subjects over uninfected persons. Ten of the 90 peptides stimulated IFN-γ secretion in the H. pylori-infected group only, whereas two out of the 90 peptides elicited a detectable IFN-γ response in the H. pylori-uninfected subjects but no response in the H. pylori-infected group. Cytokine ELISA measurements performed using in vitro PBMC culture supernatants demonstrated significantly higher levels of TNF-α, IL-2, IL-4, IL-6, IL-10, and TGF-β1 in the H. pylori-infected subjects, whereas IL-17A expression was not related to the subjects H. pylori-infection status. Our results indicate that the human T cell responses to these 90 peptides are generally increased in actively H. pylori-infected, compared with H. pylori-naïve, subjects. This information will improve understanding of the complex immune response to H. pylori, aiding rational epitope-driven vaccine design as well as helping identify other H. pylori epitopes with potentially immunoregulatory effects.

  8. Epitope mapping by epitope excision, hydrogen/deuterium exchange, and peptide-panning techniques combined with in silico analysis.

    Science.gov (United States)

    Clementi, Nicola; Mancini, Nicasio; Criscuolo, Elena; Cappelletti, Francesca; Clementi, Massimo; Burioni, Roberto

    2014-01-01

    The fine characterization of protective B cell epitopes plays a pivotal role in the development of novel vaccines. The development of epitope-based vaccines, in fact, cannot be possible without a clear definition of the antigenic regions involved in the binding between the protective antibody (Ab) and its molecular target. To achieve this result, different epitope-mapping approaches have been widely described (Clementi et al. Drug Discov Today 18(9-10):464-471, 2013). Nowadays, the best way to characterize an Ab bound region is still the resolution of Ab-antigen (Ag) co-crystal structure. Unfortunately, the crystallization approaches are not always feasible. However, different experimental strategies aimed to predict Ab-Ag interaction and followed by in silico analysis of the results may be good surrogate approaches to achieve this result. Here, we review few experimental techniques followed by the use of "basic" informatics tools for the analysis of the results.

  9. File list: Oth.Epd.50.Epitope_tags.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.Epd.50.Epitope_tags.AllCell hg19 TFs and others Epitope tags Epidermis SRX71842...0,SRX512368,SRX512366,SRX807621,SRX512367,SRX512372,SRX512373,SRX807620 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Oth.Epd.50.Epitope_tags.AllCell.bed ...

  10. File list: Oth.Unc.20.Epitope_tags.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.Unc.20.Epitope_tags.AllCell sacCer3 TFs and others Epitope tags Unclassified ht...tp://dbarchive.biosciencedbc.jp/kyushu-u/sacCer3/assembled/Oth.Unc.20.Epitope_tags.AllCell.bed ...

  11. File list: Oth.CeL.20.Epitope_tags.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.CeL.20.Epitope_tags.AllCell dm3 TFs and others Epitope tags Cell line SRX099638...099636 http://dbarchive.biosciencedbc.jp/kyushu-u/dm3/assembled/Oth.CeL.20.Epitope_tags.AllCell.bed ...

  12. File list: Oth.Unc.50.Epitope_tags.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.Unc.50.Epitope_tags.AllCell sacCer3 TFs and others Epitope tags Unclassified ht...tp://dbarchive.biosciencedbc.jp/kyushu-u/sacCer3/assembled/Oth.Unc.50.Epitope_tags.AllCell.bed ...

  13. File list: Oth.Adl.10.Epitope_tags.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.Adl.10.Epitope_tags.AllCell dm3 TFs and others Epitope tags Adult SRX181427,SRX...ttp://dbarchive.biosciencedbc.jp/kyushu-u/dm3/assembled/Oth.Adl.10.Epitope_tags.AllCell.bed ...

  14. File list: Oth.Prs.05.Epitope_tags.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.Prs.05.Epitope_tags.AllCell hg19 TFs and others Epitope tags Prostate SRX084528...,SRX084527,SRX084524 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Oth.Prs.05.Epitope_tags.AllCell.bed ...

  15. File list: Oth.PSC.20.Epitope_tags.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.PSC.20.Epitope_tags.AllCell hg19 TFs and others Epitope tags Pluripotent stem c...ell SRX555489 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Oth.PSC.20.Epitope_tags.AllCell.bed ...

  16. File list: Oth.Kid.10.Epitope_tags.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.Kid.10.Epitope_tags.AllCell hg19 TFs and others Epitope tags Kidney SRX065541,S...RX644719,SRX170375,SRX644723 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Oth.Kid.10.Epitope_tags.AllCell.bed ...

  17. File list: Oth.Brs.10.Epitope_tags.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.Brs.10.Epitope_tags.AllCell hg19 TFs and others Epitope tags Breast SRX667411,S...p://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Oth.Brs.10.Epitope_tags.AllCell.bed ...

  18. File list: Oth.EmF.50.Epitope_tags.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.EmF.50.Epitope_tags.AllCell mm9 TFs and others Epitope tags Embryonic fibroblas...RX255460,SRX204644,SRX542102,SRX204643,SRX204642 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Oth.EmF.50.Epitope_tags.AllCell.bed ...

  19. File list: Oth.ALL.50.Epitope_tags.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.ALL.50.Epitope_tags.AllCell sacCer3 TFs and others Epitope tags All cell types ...493939 http://dbarchive.biosciencedbc.jp/kyushu-u/sacCer3/assembled/Oth.ALL.50.Epitope_tags.AllCell.bed ...

  20. File list: Oth.Bld.50.Epitope_tags.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.Bld.50.Epitope_tags.AllCell mm9 TFs and others Epitope tags Blood SRX718015,SRX...,SRX180155,SRX695808 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Oth.Bld.50.Epitope_tags.AllCell.bed ...

  1. File list: Oth.Emb.05.Epitope_tags.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.Emb.05.Epitope_tags.AllCell dm3 TFs and others Epitope tags Embryo SRX066244,SR...X815533,SRX066245,SRX815531,SRX066247 http://dbarchive.biosciencedbc.jp/kyushu-u/dm3/assembled/Oth.Emb.05.Epitope_tags.AllCell.bed ...

  2. File list: Oth.CDV.20.Epitope_tags.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.CDV.20.Epitope_tags.AllCell mm9 TFs and others Epitope tags Cardiovascular SRX1...304813 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Oth.CDV.20.Epitope_tags.AllCell.bed ...

  3. File list: Oth.Emb.10.Epitope_tags.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.Emb.10.Epitope_tags.AllCell dm3 TFs and others Epitope tags Embryo SRX066244,SR...X815533,SRX066245,SRX815531,SRX066247 http://dbarchive.biosciencedbc.jp/kyushu-u/dm3/assembled/Oth.Emb.10.Epitope_tags.AllCell.bed ...

  4. File list: Oth.Neu.20.Epitope_tags.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.Neu.20.Epitope_tags.AllCell mm9 TFs and others Epitope tags Neural SRX275807,SR...SRX691799,SRX691794,SRX759286,SRX691798,SRX691797,SRX275809,SRX275811,SRX691795,SRX022866 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Oth.Neu.20.Epitope_tags.AllCell.bed ...

  5. File list: Oth.Epd.05.Epitope_tags.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.Epd.05.Epitope_tags.AllCell hg19 TFs and others Epitope tags Epidermis SRX51236...8,SRX512367,SRX718420,SRX512372,SRX512366,SRX512373,SRX807621,SRX807620 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Oth.Epd.05.Epitope_tags.AllCell.bed ...

  6. File list: Oth.NoD.50.Epitope_tags.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.NoD.50.Epitope_tags.AllCell sacCer3 TFs and others Epitope tags No description ...http://dbarchive.biosciencedbc.jp/kyushu-u/sacCer3/assembled/Oth.NoD.50.Epitope_tags.AllCell.bed ...

  7. File list: Oth.Utr.05.Epitope_tags.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.Utr.05.Epitope_tags.AllCell hg19 TFs and others Epitope tags Uterus SRX248763,S...,SRX735140,SRX735139,SRX210703,SRX210702,SRX095386,SRX968127 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Oth.Utr.05.Epitope_tags.AllCell.bed ...

  8. File list: Oth.ALL.10.Epitope_tags.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.ALL.10.Epitope_tags.AllCell sacCer3 TFs and others Epitope tags All cell types ...211370,SRX493939,SRX211371 http://dbarchive.biosciencedbc.jp/kyushu-u/sacCer3/assembled/Oth.ALL.10.Epitope_tags.AllCell.bed ...

  9. File list: Oth.Emb.50.Epitope_tags.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.Emb.50.Epitope_tags.AllCell mm9 TFs and others Epitope tags Embryo SRX485264,SR...SRX663358,SRX967653 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Oth.Emb.50.Epitope_tags.AllCell.bed ...

  10. File list: Oth.Myo.10.Epitope_tags.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.Myo.10.Epitope_tags.AllCell hg19 TFs and others Epitope tags Muscle SRX1470542,...SRX1470544 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Oth.Myo.10.Epitope_tags.AllCell.bed ...

  11. File list: Oth.Myo.20.Epitope_tags.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.Myo.20.Epitope_tags.AllCell hg19 TFs and others Epitope tags Muscle SRX1470542,...SRX1470544 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Oth.Myo.20.Epitope_tags.AllCell.bed ...

  12. File list: Oth.Neu.20.Epitope_tags.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.Neu.20.Epitope_tags.AllCell hg19 TFs and others Epitope tags Neural SRX367452,S...RX367451 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Oth.Neu.20.Epitope_tags.AllCell.bed ...

  13. File list: Oth.PSC.50.Epitope_tags.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.PSC.50.Epitope_tags.AllCell mm9 TFs and others Epitope tags Pluripotent stem ce...708,ERX320411,SRX647912,SRX204802,SRX352995 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Oth.PSC.50.Epitope_tags.AllCell.bed ...

  14. File list: Oth.Emb.20.Epitope_tags.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.Emb.20.Epitope_tags.AllCell dm3 TFs and others Epitope tags Embryo SRX815533,SR...X066244,SRX066245,SRX815531,SRX066247 http://dbarchive.biosciencedbc.jp/kyushu-u/dm3/assembled/Oth.Emb.20.Epitope_tags.AllCell.bed ...

  15. File list: Oth.Prs.20.Epitope_tags.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.Prs.20.Epitope_tags.AllCell hg19 TFs and others Epitope tags Prostate SRX084527...,SRX084528,SRX084524 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Oth.Prs.20.Epitope_tags.AllCell.bed ...

  16. File list: Oth.Brs.05.Epitope_tags.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.Brs.05.Epitope_tags.AllCell hg19 TFs and others Epitope tags Breast SRX667411,S...p://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Oth.Brs.05.Epitope_tags.AllCell.bed ...

  17. File list: Oth.NoD.50.Epitope_tags.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.NoD.50.Epitope_tags.AllCell hg19 TFs and others Epitope tags No description htt...p://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Oth.NoD.50.Epitope_tags.AllCell.bed ...

  18. File list: Oth.ALL.05.Epitope_tags.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.ALL.05.Epitope_tags.AllCell mm9 TFs and others Epitope tags All cell types SRX1...460,ERX320411,SRX695808 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Oth.ALL.05.Epitope_tags.AllCell.bed ...

  19. File list: Oth.NoD.10.Epitope_tags.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.NoD.10.Epitope_tags.AllCell sacCer3 TFs and others Epitope tags No description ...http://dbarchive.biosciencedbc.jp/kyushu-u/sacCer3/assembled/Oth.NoD.10.Epitope_tags.AllCell.bed ...

  20. File list: Oth.Myo.05.Epitope_tags.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.Myo.05.Epitope_tags.AllCell hg19 TFs and others Epitope tags Muscle SRX1470542,...SRX1470544 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Oth.Myo.05.Epitope_tags.AllCell.bed ...

  1. File list: Oth.Unc.10.Epitope_tags.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.Unc.10.Epitope_tags.AllCell hg19 TFs and others Epitope tags Unclassified SRX88...9798 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Oth.Unc.10.Epitope_tags.AllCell.bed ...

  2. File list: Oth.Bon.05.Epitope_tags.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.Bon.05.Epitope_tags.AllCell hg19 TFs and others Epitope tags Bone SRX065557,SRX...096356,SRX096358,SRX316960,SRX065556 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Oth.Bon.05.Epitope_tags.AllCell.bed ...

  3. File list: Oth.Liv.05.Epitope_tags.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.Liv.05.Epitope_tags.AllCell hg19 TFs and others Epitope tags Liver SRX1165095,S...RX1165103,SRX1165096,SRX1165104,SRX1165100,SRX1165101,SRX1165102,SRX1165090,SRX1165091 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Oth.Liv.05.Epitope_tags.AllCell.bed ...

  4. File list: Oth.Neu.10.Epitope_tags.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.Neu.10.Epitope_tags.AllCell hg19 TFs and others Epitope tags Neural SRX367452,S...RX367451 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Oth.Neu.10.Epitope_tags.AllCell.bed ...

  5. File list: Oth.ALL.20.Epitope_tags.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.ALL.20.Epitope_tags.AllCell hg19 TFs and others Epitope tags All cell types SRX...322539,SRX170374,SRX644727,SRX644719,SRX644723 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Oth.ALL.20.Epitope_tags.AllCell.bed ...

  6. File list: Oth.Neu.50.Epitope_tags.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.Neu.50.Epitope_tags.AllCell mm9 TFs and others Epitope tags Neural SRX275807,SR...SRX759284,SRX691794,SRX759286,SRX691798,SRX691797,SRX691795,SRX022866,SRX275809,SRX275811 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Oth.Neu.50.Epitope_tags.AllCell.bed ...

  7. File list: Oth.Pan.50.Epitope_tags.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.Pan.50.Epitope_tags.AllCell mm9 TFs and others Epitope tags Pancreas SRX747491,...SRX747492 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Oth.Pan.50.Epitope_tags.AllCell.bed ...

  8. File list: Oth.Unc.10.Epitope_tags.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.Unc.10.Epitope_tags.AllCell sacCer3 TFs and others Epitope tags Unclassified ht...tp://dbarchive.biosciencedbc.jp/kyushu-u/sacCer3/assembled/Oth.Unc.10.Epitope_tags.AllCell.bed ...

  9. File list: Oth.Lng.20.Epitope_tags.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.Lng.20.Epitope_tags.AllCell hg19 TFs and others Epitope tags Lung SRX119639,SRX...119641,SRX119640,SRX119642,SRX119638,SRX119637 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Oth.Lng.20.Epitope_tags.AllCell.bed ...

  10. File list: Oth.Emb.50.Epitope_tags.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.Emb.50.Epitope_tags.AllCell dm3 TFs and others Epitope tags Embryo SRX066244,SR...X815533,SRX066247,SRX066245,SRX815531 http://dbarchive.biosciencedbc.jp/kyushu-u/dm3/assembled/Oth.Emb.50.Epitope_tags.AllCell.bed ...

  11. File list: Oth.Bon.50.Epitope_tags.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.Bon.50.Epitope_tags.AllCell hg19 TFs and others Epitope tags Bone SRX065557,SRX...096356,SRX096358,SRX316960,SRX065556 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Oth.Bon.50.Epitope_tags.AllCell.bed ...

  12. File list: Oth.Epd.20.Epitope_tags.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.Epd.20.Epitope_tags.AllCell hg19 TFs and others Epitope tags Epidermis SRX71842...0,SRX512368,SRX512366,SRX807621,SRX512367,SRX512372,SRX512373,SRX807620 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Oth.Epd.20.Epitope_tags.AllCell.bed ...

  13. File list: Oth.CDV.20.Epitope_tags.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.CDV.20.Epitope_tags.AllCell hg19 TFs and others Epitope tags Cardiovascular SRX...096360,SRX096362 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Oth.CDV.20.Epitope_tags.AllCell.bed ...

  14. File list: Oth.Gon.20.Epitope_tags.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.Gon.20.Epitope_tags.AllCell mm9 TFs and others Epitope tags Gonad SRX153153,SRX...153152,SRX153151 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Oth.Gon.20.Epitope_tags.AllCell.bed ...

  15. File list: Oth.Neu.05.Epitope_tags.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.Neu.05.Epitope_tags.AllCell hg19 TFs and others Epitope tags Neural SRX367452,S...RX367451 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Oth.Neu.05.Epitope_tags.AllCell.bed ...

  16. In silico-accelerated identification of conserved and immunogenic variola/vaccinia T-cell epitopes

    DEFF Research Database (Denmark)

    Moise, Leonard; McMurry, Julie A; Buus, Søren;

    2009-01-01

    Epitopes shared by the vaccinia and variola viruses underlie the protective effect of vaccinia immunization against variola infection. We set out to identify a subset of cross-reactive epitopes using bioinformatics and immunological methods. Putative T-cell epitopes were computationally predicted...

  17. File list: Oth.Prs.10.Epitope_tags.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.Prs.10.Epitope_tags.AllCell hg19 TFs and others Epitope tags Prostate SRX084528...,SRX084527,SRX084524 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Oth.Prs.10.Epitope_tags.AllCell.bed ...

  18. File list: Oth.EmF.20.Epitope_tags.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.EmF.20.Epitope_tags.AllCell mm9 TFs and others Epitope tags Embryonic fibroblas...RX255459,SRX255462,SRX255460,SRX204643,SRX204642 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Oth.EmF.20.Epitope_tags.AllCell.bed ...

  19. File list: Oth.CDV.50.Epitope_tags.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.CDV.50.Epitope_tags.AllCell hg19 TFs and others Epitope tags Cardiovascular SRX...096360,SRX096362 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Oth.CDV.50.Epitope_tags.AllCell.bed ...

  20. File list: Oth.Gon.50.Epitope_tags.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.Gon.50.Epitope_tags.AllCell hg19 TFs and others Epitope tags Gonad SRX204898,SR...X204899 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Oth.Gon.50.Epitope_tags.AllCell.bed ...

  1. File list: Oth.CeL.10.Epitope_tags.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.CeL.10.Epitope_tags.AllCell dm3 TFs and others Epitope tags Cell line SRX099635...099636 http://dbarchive.biosciencedbc.jp/kyushu-u/dm3/assembled/Oth.CeL.10.Epitope_tags.AllCell.bed ...

  2. File list: Oth.Myo.20.Epitope_tags.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.Myo.20.Epitope_tags.AllCell mm9 TFs and others Epitope tags Muscle SRX344965,SR...http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Oth.Myo.20.Epitope_tags.AllCell.bed ...

  3. File list: Oth.Bld.05.Epitope_tags.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.Bld.05.Epitope_tags.AllCell mm9 TFs and others Epitope tags Blood SRX180156,SRX...,SRX180155,SRX695808 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Oth.Bld.05.Epitope_tags.AllCell.bed ...

  4. File list: Oth.Neu.50.Epitope_tags.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.Neu.50.Epitope_tags.AllCell hg19 TFs and others Epitope tags Neural SRX367452,S...RX367451 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Oth.Neu.50.Epitope_tags.AllCell.bed ...

  5. File list: Oth.Liv.20.Epitope_tags.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.Liv.20.Epitope_tags.AllCell hg19 TFs and others Epitope tags Liver SRX1165103,S...RX1165095,SRX1165100,SRX1165101,SRX1165090,SRX1165104,SRX1165102,SRX1165096,SRX1165091 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Oth.Liv.20.Epitope_tags.AllCell.bed ...

  6. File list: Oth.Kid.05.Epitope_tags.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.Kid.05.Epitope_tags.AllCell hg19 TFs and others Epitope tags Kidney SRX065541,S...RX644719,SRX527876,SRX644723 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Oth.Kid.05.Epitope_tags.AllCell.bed ...

  7. File list: Oth.NoD.20.Epitope_tags.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.NoD.20.Epitope_tags.AllCell hg19 TFs and others Epitope tags No description htt...p://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Oth.NoD.20.Epitope_tags.AllCell.bed ...

  8. File list: Oth.ALL.20.Epitope_tags.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.ALL.20.Epitope_tags.AllCell sacCer3 TFs and others Epitope tags All cell types ...211371,SRX493939,SRX381289 http://dbarchive.biosciencedbc.jp/kyushu-u/sacCer3/assembled/Oth.ALL.20.Epitope_tags.AllCell.bed ...

  9. File list: Oth.Unc.50.Epitope_tags.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  10. File list: Oth.Brs.20.Epitope_tags.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  11. File list: Oth.Utr.20.Epitope_tags.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  12. File list: Oth.Gon.05.Epitope_tags.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.Gon.05.Epitope_tags.AllCell hg19 TFs and others Epitope tags Gonad SRX204899,SR...X204898 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Oth.Gon.05.Epitope_tags.AllCell.bed ...

  13. File list: Oth.Gon.10.Epitope_tags.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  14. File list: Oth.PSC.20.Epitope_tags.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  15. File list: Oth.Prs.50.Epitope_tags.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  16. File list: Oth.Adl.05.Epitope_tags.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  17. File list: Oth.ALL.50.Epitope_tags.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  18. File list: Oth.CDV.10.Epitope_tags.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.CDV.10.Epitope_tags.AllCell hg19 TFs and others Epitope tags Cardiovascular SRX...096360,SRX096362 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Oth.CDV.10.Epitope_tags.AllCell.bed ...

  19. File list: Oth.Pan.05.Epitope_tags.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  20. File list: Oth.Gon.50.Epitope_tags.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.Gon.50.Epitope_tags.AllCell mm9 TFs and others Epitope tags Gonad SRX153153,SRX...153152,SRX153151 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Oth.Gon.50.Epitope_tags.AllCell.bed ...

  1. File list: Oth.YSt.10.Epitope_tags.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.YSt.10.Epitope_tags.AllCell sacCer3 TFs and others Epitope tags Yeast strain SR...1370,SRX493939,SRX211371 http://dbarchive.biosciencedbc.jp/kyushu-u/sacCer3/assembled/Oth.YSt.10.Epitope_tags.AllCell.bed ...

  2. File list: Oth.YSt.50.Epitope_tags.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  3. File list: Oth.NoD.20.Epitope_tags.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  4. File list: Oth.CDV.10.Epitope_tags.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.CDV.10.Epitope_tags.AllCell mm9 TFs and others Epitope tags Cardiovascular SRX1...304813 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Oth.CDV.10.Epitope_tags.AllCell.bed ...

  5. File list: Oth.Kid.50.Epitope_tags.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  6. File list: Oth.Unc.05.Epitope_tags.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.Unc.05.Epitope_tags.AllCell hg19 TFs and others Epitope tags Unclassified SRX88...9798 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Oth.Unc.05.Epitope_tags.AllCell.bed ...

  7. File list: Oth.CDV.50.Epitope_tags.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.CDV.50.Epitope_tags.AllCell mm9 TFs and others Epitope tags Cardiovascular SRX1...304813 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Oth.CDV.50.Epitope_tags.AllCell.bed ...

  8. File list: Oth.Oth.10.Epitope_tags.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  9. File list: Oth.Kid.20.Epitope_tags.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.Kid.20.Epitope_tags.AllCell hg19 TFs and others Epitope tags Kidney SRX065541,S...RX644727,SRX644719,SRX644723 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Oth.Kid.20.Epitope_tags.AllCell.bed ...

  10. File list: Oth.Myo.50.Epitope_tags.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.Myo.50.Epitope_tags.AllCell hg19 TFs and others Epitope tags Muscle SRX1470542,...SRX1470544 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Oth.Myo.50.Epitope_tags.AllCell.bed ...

  11. File list: Oth.PSC.10.Epitope_tags.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.PSC.10.Epitope_tags.AllCell hg19 TFs and others Epitope tags Pluripotent stem c...ell SRX555489 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Oth.PSC.10.Epitope_tags.AllCell.bed ...

  12. File list: Oth.Adl.50.Epitope_tags.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.Adl.50.Epitope_tags.AllCell dm3 TFs and others Epitope tags Adult SRX181419,SRX...ttp://dbarchive.biosciencedbc.jp/kyushu-u/dm3/assembled/Oth.Adl.50.Epitope_tags.AllCell.bed ...

  13. File list: Oth.Liv.10.Epitope_tags.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.Liv.10.Epitope_tags.AllCell hg19 TFs and others Epitope tags Liver SRX1165095,S...RX1165103,SRX1165100,SRX1165096,SRX1165104,SRX1165101,SRX1165090,SRX1165102,SRX1165091 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Oth.Liv.10.Epitope_tags.AllCell.bed ...

  14. File list: Oth.CeL.05.Epitope_tags.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.CeL.05.Epitope_tags.AllCell dm3 TFs and others Epitope tags Cell line SRX330995...099636 http://dbarchive.biosciencedbc.jp/kyushu-u/dm3/assembled/Oth.CeL.05.Epitope_tags.AllCell.bed ...

  15. File list: Oth.NoD.05.Epitope_tags.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.NoD.05.Epitope_tags.AllCell sacCer3 TFs and others Epitope tags No description ...http://dbarchive.biosciencedbc.jp/kyushu-u/sacCer3/assembled/Oth.NoD.05.Epitope_tags.AllCell.bed ...

  16. File list: Oth.YSt.05.Epitope_tags.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.YSt.05.Epitope_tags.AllCell sacCer3 TFs and others Epitope tags Yeast strain SR...1370,SRX211371,SRX493939 http://dbarchive.biosciencedbc.jp/kyushu-u/sacCer3/assembled/Oth.YSt.05.Epitope_tags.AllCell.bed ...

  17. File list: Oth.Oth.50.Epitope_tags.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.Oth.50.Epitope_tags.AllCell mm9 TFs and others Epitope tags Others SRX228677,SR...X228679,SRX228676,SRX228678 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Oth.Oth.50.Epitope_tags.AllCell.bed ...

  18. File list: Oth.Utr.50.Epitope_tags.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.Utr.50.Epitope_tags.AllCell hg19 TFs and others Epitope tags Uterus SRX188854,S...,SRX210703,SRX968127,SRX610673,SRX610674,SRX610672,SRX095386 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Oth.Utr.50.Epitope_tags.AllCell.bed ...

  19. File list: Oth.Adl.20.Epitope_tags.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  20. File list: Oth.Oth.05.Epitope_tags.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.Oth.05.Epitope_tags.AllCell mm9 TFs and others Epitope tags Others SRX228677,SR...X228676,SRX228679,SRX228678 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Oth.Oth.05.Epitope_tags.AllCell.bed ...

  1. File list: Oth.Dig.10.Epitope_tags.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.Dig.10.Epitope_tags.AllCell mm9 TFs and others Epitope tags Digestive tract SRX...365692 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Oth.Dig.10.Epitope_tags.AllCell.bed ...

  2. File list: Oth.Liv.50.Epitope_tags.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.Liv.50.Epitope_tags.AllCell hg19 TFs and others Epitope tags Liver SRX1165103,S...RX1165095,SRX1165100,SRX1165101,SRX1165104,SRX1165102,SRX1165090,SRX1165091,SRX1165096 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Oth.Liv.50.Epitope_tags.AllCell.bed ...

  3. File list: Oth.PSC.05.Epitope_tags.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.PSC.05.Epitope_tags.AllCell mm9 TFs and others Epitope tags Pluripotent stem ce...821,ERX320410,SRX266822,SRX352996,ERX320411 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Oth.PSC.05.Epitope_tags.AllCell.bed ...

  4. File list: Oth.Dig.20.Epitope_tags.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  5. File list: Oth.Gon.05.Epitope_tags.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.Gon.05.Epitope_tags.AllCell mm9 TFs and others Epitope tags Gonad SRX153152,SRX...153153,SRX153151 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Oth.Gon.05.Epitope_tags.AllCell.bed ...

  6. File list: Oth.ALL.05.Epitope_tags.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.ALL.05.Epitope_tags.AllCell hg19 TFs and others Epitope tags All cell types SRX...644715,SRX555489,SRX644719,SRX527876,SRX644723 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Oth.ALL.05.Epitope_tags.AllCell.bed ...

  7. File list: Oth.Myo.50.Epitope_tags.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.Myo.50.Epitope_tags.AllCell mm9 TFs and others Epitope tags Muscle SRX344965,SR...http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Oth.Myo.50.Epitope_tags.AllCell.bed ...

  8. File list: Oth.Myo.10.Epitope_tags.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.Myo.10.Epitope_tags.AllCell mm9 TFs and others Epitope tags Muscle SRX039346,SR...http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Oth.Myo.10.Epitope_tags.AllCell.bed ...

  9. File list: Oth.Myo.05.Epitope_tags.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.Myo.05.Epitope_tags.AllCell mm9 TFs and others Epitope tags Muscle SRX039346,SR...http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Oth.Myo.05.Epitope_tags.AllCell.bed ...

  10. File list: Oth.ALL.05.Epitope_tags.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.ALL.05.Epitope_tags.AllCell sacCer3 TFs and others Epitope tags All cell types ...211370,SRX211371,SRX493939 http://dbarchive.biosciencedbc.jp/kyushu-u/sacCer3/assembled/Oth.ALL.05.Epitope_tags.AllCell.bed ...

  11. File list: Oth.Bon.10.Epitope_tags.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

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  12. File list: Oth.ALL.20.Epitope_tags.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.ALL.20.Epitope_tags.AllCell mm9 TFs and others Epitope tags All cell types SRX1...802,SRX204643,SRX204642 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Oth.ALL.20.Epitope_tags.AllCell.bed ...

  13. File list: Oth.Brs.50.Epitope_tags.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.Brs.50.Epitope_tags.AllCell hg19 TFs and others Epitope tags Breast SRX667411,S...p://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Oth.Brs.50.Epitope_tags.AllCell.bed ...

  14. File list: Oth.Gon.10.Epitope_tags.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.Gon.10.Epitope_tags.AllCell mm9 TFs and others Epitope tags Gonad SRX153153,SRX...153151,SRX153152 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Oth.Gon.10.Epitope_tags.AllCell.bed ...

  15. Designing, Constructing and Immunogenic Evaluation of Polytope DNA Constructs by the Application of Hepatitis C Virus Immunodominant Epitopes in BALB/c Mouse

    Directory of Open Access Journals (Sweden)

    Arash Memarnejadian

    2009-01-01

    Full Text Available Objective: Polytope DNA vaccines, capable of focusing the cytotoxic T lymphocyte (CTLresponse on critical epitopes, represent a promising approach in HCV immunotherapy. Nevertheless,due to controversial rules governing epitope processing and the low level expression/immunogenicity of recombinant polytope peptides, designing and primary expression/immunogenicity analysis of these vaccine types should be the first consideration prior tocostly transgenic animal studies.Materials and Methods: Four HLA-A2 and H-2d restricted CTL epitopes were selected anddesigned in three appropriate sequential tandems based on epitope and proteasomal cleavagepredictions. The related nucleotide sequences were synthesized using SOEing PCRmethod and cloned into a pcDNA3.1 vector, either alone or fused to the small hepatitis B surfaceantigen (HBsAg-S gene. Following the preparation of polyclonal anti-sera, expression/secretion of polytopes was evaluated in Cos-7 cells by using immunofluorescence, Westernblot,dot blot, ELISA and RT-PCR techniques. The immunogenicity of the plasmids was alsoassessed through the delayed-type hypersensitivity (DTH assay in BALB/c mice.Results: Due to in silico designs and optimizations, the polytope products of constructedplasmids were efficiently detected in vitro through common techniques and HBsAg-S-basedparticles were shown to be secreted into the culture media (up to 30%. Moreover, all plasmidswere able to efficiently induce a positive DTH response while HBsAg-S fusion constructsindicated a significant immunopotential effect towards the incorporated mouse epitopes.Conclusion: Designed polytope constructs of this study are efficiently expressed and processed.They have the required initial potency for further immunogenicity analysis in transgenicmice.

  16. High-Throughput Tools for Characterization of Antibody Epitopes

    DEFF Research Database (Denmark)

    Christiansen, Anders

    multiple years. Taken together, the presented studies demonstrated new applications for the investigated techniques focusing on their utilization in epitope mapping. In the process, new insights were obtained into how antibodies recognize their targets in a major disease, i.e. food allergy....

  17. High-throughput epitope profiling of snake venom toxins

    DEFF Research Database (Denmark)

    Engmark, Mikael; Andersen, Mikael Rørdam; Laustsen, Andreas Hougaard

    Insight into the molecular details of polyclonal antivenom antibody specificity is a prerequisite for accurate prediction of cross-reactivity and can provide a basis for design of novel antivenoms. In this work, a highthroughput approach was applied to characterize linear elements in epitopes in ...... toxins from four African mamba and three neurotoxic cobra snakes obtained from public databases....

  18. Epitope Mapping of Antigenic MUC1 Peptides to Breast Cancer Antibody Fragment B27.29: A Heteronuclear NMR Study

    Energy Technology Data Exchange (ETDEWEB)

    Grinstead, Jeffrey S.; Schuman, Jason T.; Campbell, Ann P.

    2003-11-13

    MUC1 mucin is a breast cancer-associated transmembrane glycoprotein, of which the extracellular domain is formed by the repeating 20-amino acid sequence GVTSAPDTRPAPGSTAPPAH. In neoplastic breast tissue, the highly immunogenic sequence PDTRPAP (in bold above) is exposed. Antibodies raised directly against MUC1-expressing tumors offer unique access to this neoplastic state, as they represent immunologically relevant ''reverse templates'' of the tumor-associated mucin. In a previous study [Grinstead, J. S., et al. (2002) Biochemistry 41, 9946-9961], 1H NMR methods were used to correlate the effects of cryptic glycosylation outside of the PDTRPAP core epitope sequence on the recognition and binding of Mab B27.29, a monoclonal antibody raised against breast tumor cells. In the study presented here, isotope-edited NMR methods, including 15N and 13C relaxation measurements, were used to probe the recognition and binding of the PDTRPAP epitope sequence to Fab B27.29. Two peptides were studied: a one-repeat MUC1 16mer peptide of the sequence GVTSAPDTRPAPGSTA and a two-repeat MUC1 40mer peptide of the sequence (VTSAPDTRPAPGSTAPPAHG)2. 15N and 13C NMR relaxation parameters were measured for both peptides free in solution and bound to Fab B27.29. The 13CR T1 values best represent changes in the local correlation time of the peptide epitope upon binding antibody, and demonstrate that the PDTRPAP sequence is immobilized in the antibody-combining site. This result is also reflected in the appearance of the 15N- and 13C-edited HSQC spectra, where line broadening of the same peptide epitope resonances is observed. The PDTRPAP peptide epitope expands upon the peptide epitope identified previously in our group as PDTRP by homonuclear NMR experiments [Grinstead, J. S., et al. (2002) Biochemistry 41, 9946-9961], and illustrates the usefulness of the heteronuclear NMR experiments. The implications of these results are discussed within the context of MUC1 breast

  19. Differential Clade-Specific HLA-B*3501 Association with HIV-1 Disease Outcome Is Linked to Immunogenicity of a Single Gag Epitope

    Science.gov (United States)

    Matthews, Philippa C.; Koyanagi, Madoka; Harndahl, Mikkel; Stryhn, Anette; Akahoshi, Tomohiro; Gatanaga, Hiroyuki; Oka, Shinichi; Juarez Molina, Claudia; Valenzuela Ponce, Humberto; Avila Rios, Santiago; Cole, David; Carlson, Jonathan; Payne, Rebecca P.; Ogwu, Anthony; Bere, Alfred; Ndung'u, Thumbi; Gounder, Kamini; Chen, Fabian; Riddell, Lynn; Luzzi, Graz; Shapiro, Roger; Brander, Christian; Walker, Bruce; Sewell, Andrew K.; Reyes Teran, Gustavo; Heckerman, David; Hunter, Eric; Buus, Søren; Takiguchi, Masafumi; Goulder, Philip J. R.

    2012-01-01

    The strongest genetic influence on immune control in HIV-1 infection is the HLA class I genotype. Rapid disease progression in B-clade infection has been linked to HLA-B*35 expression, in particular to the less common HLA-B*3502 and HLA-B*3503 subtypes but also to the most prevalent subtype, HLA-B*3501. In these studies we first demonstrated that whereas HLA-B*3501 is associated with a high viral set point in two further B-clade-infected cohorts, in Japan and Mexico, this association does not hold in two large C-clade-infected African cohorts. We tested the hypothesis that clade-specific differences in HLA associations with disease outcomes may be related to distinct targeting of critical CD8+ T-cell epitopes. We observed that only one epitope was significantly targeted differentially, namely, the Gag-specific epitope NPPIPVGDIY (NY10, Gag positions 253 to 262) (P = 2 × 10−5). In common with two other HLA-B*3501-restricted epitopes, in Gag and Nef, that were not targeted differentially, a response toward NY10 was associated with a significantly lower viral set point. Nonimmunogenicity of NY10 in B-clade-infected subjects derives from the Gag-D260E polymorphism present in ∼90% of B-clade sequences, which critically reduces recognition of the Gag NY10 epitope. These data suggest that in spite of any inherent HLA-linked T-cell receptor repertoire differences that may exist, maximizing the breadth of the Gag-specific CD8+ T-cell response, by the addition of even a single epitope, may be of overriding importance in achieving immune control of HIV infection. This distinction is of direct relevance to development of vaccines designed to optimize the anti-HIV CD8+ T-cell response in all individuals, irrespective of HLA type. PMID:22973023

  20. Conversion of Tumors into Autologous Vaccines by Intratumoral Injection of α-Gal Glycolipids that Induce Anti-Gal/α-Gal Epitope Interaction

    Directory of Open Access Journals (Sweden)

    Uri Galili

    2011-01-01

    Full Text Available Anti-Gal is the most abundant antibody in humans, constituting 1% of immunoglobulins. Anti-Gal binds specifically α-gal epitopes (Galα1-3Galβ1-4GlcNAc-R. Immunogenicity of autologous tumor associated antigens (TAA is greatly increased by manipulating tumor cells to express α-gal epitopes and bind anti-Gal. Glycolipids with αgal epitopes (α-gal glycolipids injected into tumors insert into the tumor cell membrane. Anti-Gal binding to the multiple α-gal epitopes de novo presented on the tumor cells results in targeting of these cells to APC via the interaction between the Fc portion of the bound anti-Gal and Fcγ; receptors on APC. The APC process and present immunogenic TAA peptides and thus, effectively activate tumor specific CD4+ helper T cells and CD8+ cytotoxic T cells which destroy tumor cells in micrometastases. The induced immune response is potent enough to overcome immunosuppression by Treg cells. A phase I clinical trial indicated that α-gal glycolipid treatment has no adverse effects. In addition to achieving destruction of micrometastases in cancer patients with advance disease, α-gal glycolipid treatment may be effective as neo-adjuvant immunotherapy. Injection of α-gal glycolipids into primary tumors few weeks prior to resection can induce a protective immune response capable of destroying micrometastases expressing autologous TAA, long after primary tumor resection.

  1. Immunoinformatics study on highly expressed Mycobacterium tuberculosis genes during infection.

    Science.gov (United States)

    Nguyen Thi, Le Thuy; Sarmiento, Maria Elena; Calero, Romel; Camacho, Frank; Reyes, Fatima; Hossain, Md Murad; Gonzalez, Gustavo Sierra; Norazmi, Mohd Nor; Acosta, Armando

    2014-09-01

    The most important targets for vaccine development are the proteins that are highly expressed by the microorganisms during infection in-vivo. A number of Mycobacterium tuberculosis (Mtb) proteins are also reported to be expressed in-vivo at different phases of infection. In the present study, we analyzed multiple published databases of gene expression profiles of Mtb in-vivo at different phases of infection in animals and humans and selected 38 proteins that are highly expressed in the active, latent and reactivation phases. We predicted T- and B-cell epitopes from the selected proteins using HLAPred for T-cell epitope prediction and BCEPred combined with ABCPred for B-cell epitope prediction. For each selected proteins, regions containing both T- and B-cell epitopes were identified which might be considered as important candidates for vaccine design against tuberculosis.

  2. Expression

    Directory of Open Access Journals (Sweden)

    Wang-Xia Wang

    2014-02-01

    Full Text Available The miR-15/107 family comprises a group of 10 paralogous microRNAs (miRNAs, sharing a 5′ AGCAGC sequence. These miRNAs have overlapping targets. In order to characterize the expression of miR-15/107 family miRNAs, we employed customized TaqMan Low-Density micro-fluid PCR-array to investigate the expression of miR-15/107 family members, and other selected miRNAs, in 11 human tissues obtained at autopsy including the cerebral cortex, frontal cortex, primary visual cortex, thalamus, heart, lung, liver, kidney, spleen, stomach and skeletal muscle. miR-103, miR-195 and miR-497 were expressed at similar levels across various tissues, whereas miR-107 is enriched in brain samples. We also examined the expression patterns of evolutionarily conserved miR-15/107 miRNAs in three distinct primary rat brain cell preparations (enriched for cortical neurons, astrocytes and microglia, respectively. In primary cultures of rat brain cells, several members of the miR-15/107 family are enriched in neurons compared to other cell types in the central nervous system (CNS. In addition to mature miRNAs, we also examined the expression of precursors (pri-miRNAs. Our data suggested a generally poor correlation between the expression of mature miRNAs and their precursors. In summary, we provide a detailed study of the tissue and cell type-specific expression profile of this highly expressed and phylogenetically conserved family of miRNA genes.

  3. Developmentally regulated epitopes of cell surface arabinogalactan proteins and their relation to root tissue pattern formation.

    Science.gov (United States)

    Knox, J P; Linstead, P J; Peart, J; Cooper, C; Roberts, K

    1991-11-01

    Two polymorphic forms of an extracellular arabinogalactan protein (AGP1 and AGP2), obtained from the conditioned media of two carrot suspension-cultured cell lines, have been identified in terms of binding of the anti-plasma membrane antibodies JIM4 and MAC207. AGP1 and AGP2 have been used as immunogens to generate further anti-AGP monoclonal antibodies. JIM14 identified an epitope carried by AGP2 and also by glycoproteins of low molecular weight localized to the plant cell wall. In addition, further antibodies (JIM13 and JIM15) identified carbohydrate epitopes of the AGPs that also occur on plasma membrane glycoproteins and are expressed by patterns of cells that reflect cell position at the carrot root apex. Indirect immunofluorescence microscopy indicated that JIM13 recognized the surface of cells forming the epidermis and cells marking the region and axis of the future xylem. JIM15 recognized a pattern of cells directly complementary to the JIM13 pattern. The panel of anti-AGP monoclonal antibodies now available indicates groups of cells within the root meristem that may reflect an early pre-pattern of the tissues of the mature root structure and suggests extensive modulation of cell surface AGPs during cell development and the positioning of cells within the apex.

  4. Production and characterization of monoclonal antibody specific to recombinant dengue multi-epitope protein.

    Science.gov (United States)

    Abhyankar, Ajay Vinayak; Bhargava, Rakesh; Jana, Asha Mukul; Sahni, Ajay Kumar; Rao, P V Lakshmana

    2008-06-01

    Monoclonal antibodies against novel dengue recombinant protein were produced following immunization of Balb/c mice with recombinant dengue multi-epitope protein (r-DMEP) expressed in Escherichia coli vector and purified in a single-step chromatography system. Antigenicity of r-DMEP was evaluated by dot enzyme immunoassay. Mice were immunized intraperitoneally with five doses each of 100 microg of this novel antigen at 1-week intervals and a final intravenous booster dose prior to the fusion. Hybridomas resulted from fusion of myeloma cells and splenocytes using PEG-1500 as an additive. Selection of the hybrids was done using HAT medium, and the hybrids thus selected were finally screened qualitatively and quantitatively by dot and plate immunoassays, respectively. Five antibody secretory hybrid clones exhibited specific reactivity against r-DMEP by dot-ELISA, whereas a lone clone was found to be cross-reactive with Japanese encephalitis virus (JEV). Monoclonal antibodies (MAbs) specific to r-DME protein recognized the envelope and non-structural epitopes by Western blot analysis. These MAbs were further checked for their diagnostic efficacy using dengue suspected clinical samples and found overall sensitivity and specificity for DRDE dipstick ELISA. MAb-based dipstick ELISA results were 85%, 75% and 85%, 90%, respectively.

  5. Two highly similar LAEDDTNAQKT and LTDKIGTEI epitopes in G glycoprotein may be useful for effective epitope based vaccine design against pathogenic Henipavirus.

    Science.gov (United States)

    Parvege, Md Masud; Rahman, Monzilur; Nibir, Yead Morshed; Hossain, Mohammad Shahnoor

    2016-04-01

    Nipah virus and Hendra virus, two members of the genus Henipavirus, are newly emerging zoonotic pathogens which cause acute respiratory illness and severe encephalitis in human. Lack of the effective antiviral therapy endorses the urgency for the development of vaccine against these deadly viruses. In this study, we employed various computational approaches to identify epitopes which has the potential for vaccine development. By analyzing the immune parameters of the conserved sequences of G glycoprotein using various databases and bioinformatics tools, we identified two potential epitopes which may be used as peptide vaccines. Using different B cell epitope prediction servers, four highly similar B cell epitopes were identified. Immunoinformatics analyses revealed that LAEDDTNAQKT is a highly flexible and accessible B-cell epitope to antibody. Highly similar putative CTL epitopes were analyzed for their binding with the HLA-C 12*03 molecule. Docking simulation assay revealed that LTDKIGTEI has significantly lower binding energy, which bolstered its potential as epitope-based vaccine design. Finally, cytotoxicity analysis has also justified their potential as promising epitope-based vaccine candidate. In sum, our computational analysis indicates that either LAEDDTNAQKT or LTDKIGTEI epitope holds a promise for the development of universal vaccine against all kinds of pathogenic Henipavirus. Further in vivo and in vitro studies are necessary to validate the obtained findings.

  6. MIMOX: a web tool for phage display based epitope mapping

    Directory of Open Access Journals (Sweden)

    Honda Wataru

    2006-10-01

    Full Text Available Abstract Background Phage display is widely used in basic research such as the exploration of protein-protein interaction sites and networks, and applied research such as the development of new drugs, vaccines, and diagnostics. It has also become a promising method for epitope mapping. Research on new algorithms that assist and automate phage display based epitope mapping has attracted many groups. Most of the existing tools have not been implemented as an online service until now however, making it less convenient for the community to access, utilize, and evaluate them. Results We present MIMOX, a free web tool that helps to map the native epitope of an antibody based on one or more user supplied mimotopes and the antigen structure. MIMOX was coded in Perl using modules from the Bioperl project. It has two sections. In the first section, MIMOX provides a simple interface for ClustalW to align a set of mimotopes. It also provides a simple statistical method to derive the consensus sequence and embeds JalView as a Java applet to view and manage the alignment. In the second section, MIMOX can map a single mimotope or a consensus sequence of a set of mimotopes, on to the corresponding antigen structure and search for all of the clusters of residues that could represent the native epitope. NACCESS is used to evaluate the surface accessibility of the candidate clusters; and Jmol is embedded to view them interactively in their 3D context. Initial case studies show that MIMOX can reproduce mappings from existing tools such as FINDMAP and 3DEX, as well as providing novel, rational results. Conclusion A web-based tool called MIMOX has been developed for phage display based epitope mapping. As a publicly available online service in this area, it is convenient for the community to access, utilize, and evaluate, complementing other existing programs. MIMOX is freely available at http://web.kuicr.kyoto-u.ac.jp/~hjian/mimox.

  7. HLA-A2–Restricted Cytotoxic T Lymphocyte Epitopes from Human Heparanase as Novel Targets for Broad-Spectrum Tumor Immunotherapy

    Directory of Open Access Journals (Sweden)

    Ting Chen

    2008-09-01

    Full Text Available Peptide vaccination for cancer immunotherapy requires identification of peptide epitopes derived from antigenic proteins associated with tumors. Heparanase (Hpa is broadly expressed in various advanced tumors and seems to be an attractive new tumor-associated antigen. The present study was designed to predict and identify HLA-A2– restricted cytotoxic T lymphocyte (CTL epitopes in the protein of human Hpa. For this purpose, HLA-A2–restricted CTL epitopes were identified using the following four-step procedure: 1 a computer-based epitope prediction from the amino acid sequence of human Hpa, 2 a peptide-binding assay to determine the affinity of the predicted protein with the HLA-A2 molecule, 3 stimulation of the primary T-cell response against the predicted peptides in vitro, and 4 testing of the induced CTLs toward different kinds of carcinoma cells expressing Hpa antigens and/or HLA-A2. The results demonstrated that, of the tested peptides, effectors induced by peptides of human Hpa containing residues 525-533 (PAFSYSFFV, Hpa525, 277-285 (KMLKSFLKA, Hpa277, and 405-413 (WLSLLFKKL, Hpa405 could effectively lyse various tumor cell lines that were Hpa-positive and HLA-A2-matched. We also found that these peptide-specific CTLs could not lyse autologous lymphocytes with low Hpa activity. Further study revealed that Hpa525, Hpa277, and Hpa405 peptides increased the frequency of IFN-γ–producing T cells compared to a negative peptide. Our results suggest that Hpa525, Hpa277, and Hpa405 peptides are new HLA-A2–restricted CTL epitopes capable of inducing Hpa-specific CTLs in vitro. Because Hpa is expressed in most advanced malignant tumors, Hpa525, Hpa277, and Hpa405 peptide–based vaccines may be useful for the immunotherapy for patients with advanced tumors.

  8. Synthetic B-Cell Epitopes Eliciting Cross-Neutralizing Antibodies: Strategies for Future Dengue Vaccine

    Science.gov (United States)

    Poh, Chit Laa; Kirk, Kristin; McBride, William John Hannan; Aaskov, John; Grollo, Lara

    2016-01-01

    Dengue virus (DENV) is a major public health threat worldwide. A key element in protection from dengue fever is the neutralising antibody response. Anti-dengue IgG purified from DENV-2 infected human sera showed reactivity against several peptides when evaluated by ELISA and epitope extraction techniques. A multi-step computational approach predicted six antigenic regions within the E protein of DENV-2 that concur with the 6 epitopes identified by the combined ELISA and epitope extraction approach. The selected peptides representing B-cell epitopes were attached to a known dengue T-helper epitope and evaluated for their vaccine potency. Immunization of mice revealed two novel synthetic vaccine constructs that elicited good humoral immune responses and produced cross-reactive neutralising antibodies against DENV-1, 2 and 3. The findings indicate new directions for epitope mapping and contribute towards the future development of multi-epitope based synthetic peptide vaccine. PMID:27223692

  9. Epitope-specific antibody levels in tuberculosis: biomarkers of protection, disease and response to treatment.

    Directory of Open Access Journals (Sweden)

    Graham H Bothamley

    2014-06-01

    Full Text Available Monoclonal antibodies restricted to Mycobacterium tuberculosis can measure epitope-specific antibody levels in a competition assay. Immunodominant epitopes were defined from clinical samples and related to the clinical spectrum of disease. Antibody to the immunodominant epitopes was associated with HLA-DR15. Occupational exposure showed a different response and was consistent with recognition of dormancy related proteins and protection despite exposure to tuberculosis. Studies in leprosy revealed the importance of immune deviation and the relationships between T and B cell epitopes. During treatment, antibody levels increased, epitope spreading occurred, but the affinity constants remained the same after further antigen exposure, suggesting constraints on the process of epitope selection. Epitope-specific antibody levels have a potential role as biomarkers for new vaccines which might prevent the progression of latent to active tuberculosis and as tools to measure treatment effects on subpopulations of tubercle bacilli.

  10. Synthetic B-Cell Epitopes Eliciting Cross-Neutralizing Antibodies: Strategies for Future Dengue Vaccine.

    Directory of Open Access Journals (Sweden)

    Babu Ramanathan

    Full Text Available Dengue virus (DENV is a major public health threat worldwide. A key element in protection from dengue fever is the neutralising antibody response. Anti-dengue IgG purified from DENV-2 infected human sera showed reactivity against several peptides when evaluated by ELISA and epitope extraction techniques. A multi-step computational approach predicted six antigenic regions within the E protein of DENV-2 that concur with the 6 epitopes identified by the combined ELISA and epitope extraction approach. The selected peptides representing B-cell epitopes were attached to a known dengue T-helper epitope and evaluated for their vaccine potency. Immunization of mice revealed two novel synthetic vaccine constructs that elicited good humoral immune responses and produced cross-reactive neutralising antibodies against DENV-1, 2 and 3. The findings indicate new directions for epitope mapping and contribute towards the future development of multi-epitope based synthetic peptide vaccine.

  11. Synthetic B-Cell Epitopes Eliciting Cross-Neutralizing Antibodies: Strategies for Future Dengue Vaccine.

    Science.gov (United States)

    Ramanathan, Babu; Poh, Chit Laa; Kirk, Kristin; McBride, William John Hannan; Aaskov, John; Grollo, Lara

    2016-01-01

    Dengue virus (DENV) is a major public health threat worldwide. A key element in protection from dengue fever is the neutralising antibody response. Anti-dengue IgG purified from DENV-2 infected human sera showed reactivity against several peptides when evaluated by ELISA and epitope extraction techniques. A multi-step computational approach predicted six antigenic regions within the E protein of DENV-2 that concur with the 6 epitopes identified by the combined ELISA and epitope extraction approach. The selected peptides representing B-cell epitopes were attached to a known dengue T-helper epitope and evaluated for their vaccine potency. Immunization of mice revealed two novel synthetic vaccine constructs that elicited good humoral immune responses and produced cross-reactive neutralising antibodies against DENV-1, 2 and 3. The findings indicate new directions for epitope mapping and contribute towards the future development of multi-epitope based synthetic peptide vaccine.

  12. Identification of T-cell epitopes of Lol p 9, a major allergen of ryegrass (Lolium perenne) pollen.

    Science.gov (United States)

    Blaher, B; Suphioglu, C; Knox, R B; Singh, M B; McCluskey, J; Rolland, J M

    1996-07-01

    T-cell recognition of Lol p 9, a major allergen of ryegrass pollen, was investigated by using a T-cell line and T-cell clones generated from the peripheral blood of an atopic donor. The T-cell line reacted with purified Lol p 9, as well as with crude ryegrass pollen extract, but failed to cross-react with Bermuda grass pollen extract. All of six T-cell clones generated from this line proliferated in response to Lol p 9. Epitope mapping was carried out with a panel of 34 overlapping synthetic peptides, which spanned the entire sequence of the Lol p 9 12R isoform. The T-cell line responded to two of the peptides, Lol p 9 (105-116) and Lol p 9 (193-204), whereas reactivity with one or other of these peptides was shown by five T-cell clones. These two peptides contained sequences consistent with motifs previously reported for major histocompatibility complex class II-restricted peptides. HLA antibody blocking studies showed that presentation of peptide Lol p 9 (105-116) to one T-cell clone was HLA-DR-restricted; this clone expressed a T helper cell phenotype (CD3+, CD4+) and the T-cell receptor alpha beta. The identification of immunodominant T-cell epitope(s) on allergens is essential for devising safer and more effective immunotherapy strategies, which can interrupt the chain of events leading to allergic disease.

  13. Isolating subpopulations of human epidermal basal cells based on polyclonal serum against trypsin-resistant CSPG4 epitopes.

    Science.gov (United States)

    Gunnarsson, Anders Patrik; Christensen, Rikke; Praetorius, Jeppe; Jensen, Uffe Birk

    2017-01-15

    Chondroitin sulfate proteoglycan 4 (CSPG4) is highly expressed by human epidermal keratinocytes located at the tip of the dermal papilla where keratinocytes show characteristics of stem cells. However, since available antibodies to CSPG4 are directed against trypsin-sensitive epitopes we have been unable to study these keratinocytes isolated directly from skin samples by flow cytometry. By choosing epitopes of CSPG4 relatively close to the cell membrane we were able to generate a polyclonal antibody that successfully detects CSPG4 on keratinocytes after trypsinization. Although CSPG4-positive basal cells express higher levels of Itgβ1 the colony-forming efficiency is slightly lower than CSPG4-negative basal cells. Sorting the directly isolated keratinocytes based on Itgβ1 did not reveal differences in colony-forming efficiency between keratinocytes expressing high or low levels of Itgβ1. However, after the first passage Itgβ1 could be used to predict colony-forming efficiency whether the culture was established from CSPG4-positive or CSPG4-negative basal cell keratinocytes. Although we were unable to detect differences in the colony-forming assay, global gene expression profiling showed that CSPG4-positive basal cell keratinocytes are distinct from CSPG4-negative basal cell keratinocytes. Our study demonstrates that it is possible to generate antibodies against trypsin-resistant epitopes of CSPG4. Our study also documents a marked change in behaviour upon cell culturing and challenges the way we assess for stemness within the human epidermal basal layer.

  14. Automated Detection of Conformational Epitopes Using Phage Display Peptide Sequences

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    Surendra S Negi

    2009-01-01

    Full Text Available Background: Precise determination of conformational epitopes of neutralizing antibodies represents a key step in the rational design of novel vaccines. A powerful experimental method to gain insights on the physical chemical nature of conformational epitopes is the selection of linear peptides that bind with high affinities to a monoclonal antibody of interest by phage display technology. However, the structural characterization of conformational epitopes from these mimotopes is not straightforward, and in the past the interpretation of peptide sequences from phage display experiments focused on linear sequence analysis to find a consensus sequence or common sequence motifs.Results: We present a fully automated search method, EpiSearch that predicts the possible location of conformational epitopes on the surface of an antigen. The algorithm uses peptide sequences from phage display experiments as input, and ranks all surface exposed patches according to the frequency distribution of similar residues in the peptides and in the patch. We have tested the performance of the EpiSearch algorithm for six experimental data sets of phage display experiments, the human epidermal growth factor receptor-2 (HER-2/neu, the antibody mAb Bo2C11 targeting the C2 domain of FVIII, antibodies mAb 17b and mAb b12 of the HIV envelope protein gp120, mAb 13b5 targeting HIV-1 capsid protein and 80R of the SARS coronavirus spike protein. In all these examples the conformational epitopes as determined by the X-ray crystal structures of the antibody-antigen complexes, were found within the highest scoring patches of EpiSearch, covering in most cases more than 50% residues of experimental observed conformational epitopes. Input options of the program include mapping of a single peptide or a set of peptides on the antigen structure, and the results of the calculation can be visualized on our interactive web server.Availability: Users can access the EpiSearch from our web

  15. Epitope mapping porcine reproductive and respiratory syndrome virus by phage display: the nsp2 fragment of the replicase polyprotein contains a cluster of B-cell epitopes

    DEFF Research Database (Denmark)

    Oleksiewicz, M.B.; Bøtner, Anette; Toft, P.;

    2001-01-01

    We screened phage display libraries of porcine reproductive and respiratory syndrome virus (PRRSV) protein fragments with sera from experimentally infected pigs to identify linear B-cell epitopes that are commonly recognized during infection in vivo. We identified 10 linear epitope sites (ES) 11...

  16. Three Candidate Epitope-Vaccines in Combination Inducing High Levels of Multiantibodies Against HIV-1

    Institute of Scientific and Technical Information of China (English)

    刘祖强; 田海军; 王颖; 陈应华

    2003-01-01

    HIV-1 mutation results in immune evasion, which presents a serious challenge for conventional strategies for developing effective vaccines.So far, much experimental evidence indicates that HIV-1 particles in the blood of patients can be cleaned principally by neutralizing antibodies.Based on these facts, we prepared triple combination of epitope-vaccines with the objective of inducing antibodies with predefined multi-epitope-specificity against HIV-1.According to the sequences of three neutralizing epitopes (RILAVERYLKD, ELDKWA and GPGRAFY, designated E1, E2, and E3, respectively) on HIV-1 envelope proteins, three epitope-peptides ((E1)2: C-(RILAVERYLKDG)2; (E2)4: C-(ELDKWAG)4; and (E3)2: C-(GPGRAFY)2) were synthesized and then conjugated with carrier protein keyhole limpet hemocyanin (KLH) or bovine serum albumin (BSA), and used for immunizing rabbits.After the vaccine course, the triple combination of epitope-vaccines induced high levels of predefined multi-epitope-specific antibodies.An immunoblotting-analysis demonstrated that the antibodies could recognize the native epitopes on both gp41 protein and V3 loop peptide.Furthermore, we compared the immune responses of three doses of epitope-peptides in the candidate epitope-vaccine.Strong antibody responses to three epitopes were observed in a dose dependent manner, with increasing dose raising the immune response.This result indicated that immunotolerance did not occur using an epitope vaccine dose of 80 μg.Thus, our results demonstrate that epitope-vaccines in combination can synchronously induce high levels of antibodies with predefined multi-epitope-specificity against HIV-1, and may be used to develop effective vaccines against HIV as a new strategy.

  17. Anti-epitope antibody,a novel site-directed antibody against human acetylcholinesterase

    Institute of Scientific and Technical Information of China (English)

    Xing-mei ZHANG; Gang LIU; Man-ji SUN

    2004-01-01

    AIM: To construct synthetic antigens using the epitope of human brain acetylcholinesterase (hbAChE) for induction and detection of the specific antibody against the epitope, and to analyse the immunogenicity of the antibody.METHODS: The epitope (RTVLVSMNYR, amino acids 143-152) of hbAChE was chemically synthesized, coupled with the carrier protein keyhole limpet hemocyanin (KLH) to construct an artificial immunogen (KLH-epitope), and injected into rabbits to raise antibody. The epitope conjugated with bovine serum albumin (BSA) was used as the detection antigen. The specificity of the antibody was tested by enzyme-linked immunosorbent assay (ELISA) and Western blotting. The immunoreaction between the anti-recombinant human butyrylcholinesterase (rhBChE)polyclonal antibody and the biotinylated-epitope was examined by indirect ELISA. RESULTS: The erythrocyte AChE, the hbAChE, rhBChE and the BSA-epitope all immunoreacted with the anti-epitope antibody against the epitope (143-152) of hbAChE, whereas the torpedo AChE did not. CONCLUSION: The hbAChE, the human erythrocyte AChE and hBChE share the conservative antigenic epitope RTVLVSMNYR, hence they can all immunoreact with the anti-epitope antibody. Since the epitope of hbAChE is less similar with the aligned amino acid sequences of AChE of Torpedo californica or Torpedo marmorata, there is not any immunoreactivity between them. The R, M, and N residues in the epitope seem to be necessary radicals for the conservation of antigenicity.

  18. 'Multi-epitope-targeted' immune-specific therapy for a multiple sclerosis-like disease via engineered multi-epitope protein is superior to peptides.

    Directory of Open Access Journals (Sweden)

    Nathali Kaushansky

    Full Text Available Antigen-induced peripheral tolerance is potentially one of the most efficient and specific therapeutic approaches for autoimmune diseases. Although highly effective in animal models, antigen-based strategies have not yet been translated into practicable human therapy, and several clinical trials using a single antigen or peptidic-epitope in multiple sclerosis (MS yielded disappointing results. In these clinical trials, however, the apparent complexity and dynamics of the pathogenic autoimmunity associated with MS, which result from the multiplicity of potential target antigens and "epitope spread", have not been sufficiently considered. Thus, targeting pathogenic T-cells reactive against a single antigen/epitope is unlikely to be sufficient; to be effective, immunospecific therapy to MS should logically neutralize concomitantly T-cells reactive against as many major target antigens/epitopes as possible. We investigated such "multi-epitope-targeting" approach in murine experimental autoimmune encephalomyelitis (EAE associated with a single ("classical" or multiple ("complex" anti-myelin autoreactivities, using cocktail of different encephalitogenic peptides vis-a-vis artificial multi-epitope-protein (designated Y-MSPc encompassing rationally selected MS-relevant epitopes of five major myelin antigens, as "multi-epitope-targeting" agents. Y-MSPc was superior to peptide(s in concomitantly downregulating pathogenic T-cells reactive against multiple myelin antigens/epitopes, via inducing more effective, longer lasting peripheral regulatory mechanisms (cytokine shift, anergy, and Foxp3+ CTLA4+ regulatory T-cells. Y-MSPc was also consistently more effective than the disease-inducing single peptide or peptide cocktail, not only in suppressing the development of "classical" or "complex EAE" or ameliorating ongoing disease, but most importantly, in reversing chronic EAE. Overall, our data emphasize that a "multi-epitope-targeting" strategy is required for

  19. A General Method to Discover Epitopes from Sera.

    Directory of Open Access Journals (Sweden)

    Kurt Whittemore

    Full Text Available Antigen-antibody complexes are central players in an effective immune response. However, finding those interactions relevant to a particular disease state can be arduous. Nonetheless many paths to discovery have been explored since deciphering these interactions can greatly facilitate the development of new diagnostics, therapeutics, and vaccines. In silico B cell epitope mapping approaches have been widely pursued, though success has not been consistent. Antibody mixtures in immune sera have been used as handles for biologically relevant antigens, but these and other experimental approaches have proven resource intensive and time consuming. In addition, these methods are often tailored to individual diseases or a specific proteome, rather than providing a universal platform. Most of these methods are not able to identify the specific antibody's epitopes from unknown antigens, such as un-annotated neo antigens in cancer. Alternatively, a peptide library comprised of sequences unrestricted by naturally-found protein space provides for a universal search for mimotopes of an antibody's epitope. Here we present the utility of such a non-natural random sequence library of 10,000 peptides physically addressed on a microarray for mimotope discovery without sequence information of the specific antigen. The peptide arrays were probed with serum from an antigen-immunized rabbit, or alternatively probed with serum pre-absorbed with the same immunizing antigen. With this positive and negative screening scheme, we identified the library-peptides as the mimotopes of the antigen. The unique library peptides were successfully used to isolate antigen-specific antibodies from complete immune serum. Sequence analysis of these peptides revealed the epitopes in the immunized antigen. We present this method as an inexpensive, efficient method for identifying mimotopes of any antibody's targets. These mimotopes should be useful in defining both components of the

  20. Elicitation of structure-specific antibodies by epitope scaffolds

    OpenAIRE

    2010-01-01

    Elicitation of antibodies against targets that are immunorecessive, cryptic, or transient in their native context has been a challenge for vaccine design. Here we demonstrate the elicitation of structure-specific antibodies against the HIV-1 gp41 epitope of the broadly neutralizing antibody 2F5. This conformationally flexible region of gp41 assumes mostly helical conformations but adopts a kinked, extended structure when bound by antibody 2F5. Computational techniques were employed to transpl...

  1. Combinatorial Contextualization of Peptidic Epitopes for Enhanced Cellular Immunity

    Science.gov (United States)

    Ito, Masaki; Hayashi, Kazumi; Adachi, Eru; Minamisawa, Tamiko; Homma, Sadamu; Koido, Shigeo; Shiba, Kiyotaka

    2014-01-01

    Invocation of cellular immunity by epitopic peptides remains largely dependent on empirically developed protocols, such as interfusion of aluminum salts or emulsification using terpenoids and surfactants. To explore novel vaccine formulation, epitopic peptide motifs were co-programmed with structural motifs to produce artificial antigens using our “motif-programming” approach. As a proof of concept, we used an ovalbumin (OVA) system and prepared an artificial protein library by combinatorially polymerizing MHC class I and II sequences from OVA along with a sequence that tends to form secondary structures. The purified endotoxin-free proteins were then examined for their ability to activate OVA-specific T-cell hybridoma cells after being processed within dendritic cells. One clone, F37A (containing three MHC I and two MHC II OVA epitopes), possessed a greater ability to evoke cellular immunity than the native OVA or the other artificial antigens. The sensitivity profiles of drugs that interfered with the F37A uptake differed from those of the other artificial proteins and OVA, suggesting that alteration of the cross-presentation pathway is responsible for the enhanced immunogenicity. Moreover, F37A, but not an epitopic peptide, invoked cellular immunity when injected together with monophosphoryl lipid A (MPL), and retarded tumor growth in mice. Thus, an artificially synthesized protein antigen induced cellular immunity in vivo in the absence of incomplete Freund's adjuvant or aluminum salts. The method described here could be potentially used for developing vaccines for such intractable ailments as AIDS, malaria and cancer, ailments in which cellular immunity likely play a crucial role in prevention and treatment. PMID:25343355

  2. Common antiviral cytotoxic t-lymphocyte epitope for diverse arenaviruses.

    Science.gov (United States)

    Oldstone, M B; Lewicki, H; Homann, D; Nguyen, C; Julien, S; Gairin, J E

    2001-07-01

    Members of the Arenaviridae family have been isolated from mammalian hosts in disparate geographic locations, leading to their grouping as Old World types (i.e., lymphocytic choriomeningitis virus [LCMV], Lassa fever virus [LFV], Mopeia virus, and Mobala virus) and New World types (i.e., Junin, Machupo, Tacaribe, and Sabia viruses) (C. J. Peters, M. J. Buchmeier, P. E. Rollin, and T. G. Ksiazek, p. 1521-1551, in B. N. Fields, D. M. Knipe, and P. M. Howley [ed.], Fields virology, 3rd ed., 1996; P. J. Southern, p. 1505-1519, in B. N. Fields, D. M. Knipe, and P. M. Howley [ed.], Fields virology, 3rd ed., 1996). Several types in both groups-LFV, Junin, Machupo, and Sabia viruses-cause severe and often lethal human diseases. By sequence comparison, we noted that eight Old World and New World arenaviruses share several amino acids with the nucleoprotein (NP) that consists of amino acids (aa) 118 to 126 (NP 118-126) (RPQASGVYM) of LCMV that comprise the immunodominant cytotoxic T-lymphocyte (CTL) epitope for H-2(d) mice (32). This L(d)-restricted epitope constituted >97% of the total bulk CTLs produced in the specific antiviral or clonal responses of H-2(d) BALB mice. NP 118-126 of the Old World arenaviruses LFV, Mopeia virus, and LCMV and the New World arenavirus Sabia virus bound at high affinity to L(d). The primary H-2(d) CTL anti-LCMV response as well as that of a CTL clone responsive to LCMV NP 118-126 recognized target cells coated with NP 118-126 peptides derived from LCMV, LFV, and Mopeia virus but not Sabia virus, indicating that a common functional NP epitope exists among Old World arenaviruses. Use of site-specific amino acid exchanges in the NP CTL epitope among these arenaviruses identified amino acids involved in major histocompatibility complex binding and CTL recognition.

  3. Optimal selection of epitopes for TXP-immunoaffinity mass spectrometry

    Directory of Open Access Journals (Sweden)

    Joos Thomas

    2010-06-01

    Full Text Available Abstract Background Mass spectrometry (MS based protein profiling has become one of the key technologies in biomedical research and biomarker discovery. One bottleneck in MS-based protein analysis is sample preparation and an efficient fractionation step to reduce the complexity of the biological samples, which are too complex to be analyzed directly with MS. Sample preparation strategies that reduce the complexity of tryptic digests by using immunoaffinity based methods have shown to lead to a substantial increase in throughput and sensitivity in the proteomic mass spectrometry approach. The limitation of using such immunoaffinity-based approaches is the availability of the appropriate peptide specific capture antibodies. Recent developments in these approaches, where subsets of peptides with short identical terminal sequences can be enriched using antibodies directed against short terminal epitopes, promise a significant gain in efficiency. Results We show that the minimal set of terminal epitopes for the coverage of a target protein list can be found by the formulation as a set cover problem, preceded by a filtering pipeline for the exclusion of peptides and target epitopes with undesirable properties. Conclusions For small datasets (a few hundred proteins it is possible to solve the problem to optimality with moderate computational effort using commercial or free solvers. Larger datasets, like full proteomes require the use of heuristics.

  4. Common food allergens and their IgE-binding epitopes

    Directory of Open Access Journals (Sweden)

    Hiroaki Matsuo

    2015-10-01

    Full Text Available Food allergy is an adverse immune response to certain kinds of food. Although any food can cause allergic reactions, chicken egg, cow's milk, wheat, shellfish, fruit, and buckwheat account for 75% of food allergies in Japan. Allergen-specific immunoglobulin E (IgE antibodies play a pivotal role in the development of food allergy. Recent advances in molecular biological techniques have enabled the efficient analysis of food allergens. As a result, many food allergens have been identified, and their molecular structure and IgE-binding epitopes have also been identified. Studies of allergens have demonstrated that IgE antibodies specific to allergen components and/or the peptide epitopes are good indicators for the identification of patients with food allergy, prediction of clinical severity and development of tolerance. In this review, we summarize our current knowledge regarding the allergens and IgE epitopes in the well-researched allergies to chicken egg, cow's milk, wheat, shrimp, and peanut.

  5. Toward Effective HIV Vaccination INDUCTION OF BINARY EPITOPE REACTIVE ANTIBODIES WITH BROAD HIV NEUTRALIZING ACTIVITY

    Energy Technology Data Exchange (ETDEWEB)

    Nishiyama, Yasuhiro; Planque, Stephanie; Mitsuda, Yukie; Nitti, Giovanni; Taguchi, Hiroaki; Jin, Lei; Symersky, Jindrich; Boivin, Stephane; Sienczyk, Marcin; Salas, Maria; Hanson, Carl V.; Paul, Sudhir; (Texas-MED); (Viral Rickettsial)

    2009-11-23

    We describe murine monoclonal antibodies (mAbs) raised by immunization with an electrophilic gp120 analog (E-gp120) expressing the rare ability to neutralize genetically heterologous human immunodeficiency virus (HIV) strains. Unlike gp120, E-gp120 formed covalent oligomers. The reactivity of gp120 and E-gp120 with mAbs to reference neutralizing epitopes was markedly different, indicating their divergent structures. Epitope mapping with synthetic peptides and electrophilic peptide analogs indicated binary recognition of two distinct gp120 regions by anti-E-gp120 mAbs, the 421-433 and 288-306 peptide regions. Univalent Fab and single chain Fv fragments expressed the ability to recognize both peptides. X-ray crystallography of an anti-E-gp120 Fab fragment revealed two neighboring cavities, the typical antigen-binding cavity formed by the complementarity determining regions (CDRs) and another cavity dominated by antibody heavy chain variable (VH) domain framework (FR) residues. Substitution of the FR cavity VH Lys-19 residue by an Ala residue resulted in attenuated binding of the 421-433 region peptide probe. The CDRs and VH FR replacement/silent mutation ratios exceeded the ratio for a random mutation process, suggesting adaptive development of both putative binding sites. All mAbs studied were derived from VH1 family genes, suggesting biased recruitment of the V gene germ line repertoire by E-gp120. The conserved 421-433 region of gp120 is essential for HIV binding to host CD4 receptors. This region is recognized weakly by the FR of antibodies produced without exposure to HIV, but it usually fails to induce adaptive synthesis of neutralizing antibodies. We present models accounting for improved CD4-binding site recognition and broad HIV neutralizing activity of the mAbs, long sought goals in HIV vaccine development.

  6. CETCh-seq: CRISPR epitope tagging ChIP-seq of DNA-binding proteins.

    Science.gov (United States)

    Savic, Daniel; Partridge, E Christopher; Newberry, Kimberly M; Smith, Sophia B; Meadows, Sarah K; Roberts, Brian S; Mackiewicz, Mark; Mendenhall, Eric M; Myers, Richard M

    2015-10-01

    Chromatin immunoprecipitation followed by next-generation DNA sequencing (ChIP-seq) is a widely used technique for identifying transcription factor (TF) binding events throughout an entire genome. However, ChIP-seq is limited by the availability of suitable ChIP-seq grade antibodies, and the vast majority of commercially available antibodies fail to generate usable data sets. To ameliorate these technical obstacles, we present a robust methodological approach for performing ChIP-seq through epitope tagging of endogenous TFs. We used clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9-based genome editing technology to develop CRISPR epitope tagging ChIP-seq (CETCh-seq) of DNA-binding proteins. We assessed the feasibility of CETCh-seq by tagging several DNA-binding proteins spanning a wide range of endogenous expression levels in the hepatocellular carcinoma cell line HepG2. Our data exhibit strong correlations between both replicate types as well as with standard ChIP-seq approaches that use TF antibodies. Notably, we also observed minimal changes to the cellular transcriptome and to the expression of the tagged TF. To examine the robustness of our technique, we further performed CETCh-seq in the breast adenocarcinoma cell line MCF7 as well as mouse embryonic stem cells and observed similarly high correlations. Collectively, these data highlight the applicability of CETCh-seq to accurately define the genome-wide binding profiles of DNA-binding proteins, allowing for a straightforward methodology to potentially assay the complete repertoire of TFs, including the large fraction for which ChIP-quality antibodies are not available.

  7. Molecular Characterization of Two Monoclonal Antibodies against the Same Epitope on B-Cell Receptor Associated Protein 31

    OpenAIRE

    Kim, Won-Tae; Shin, Saemina; Hwang, Hyo Jeong; Kim, Min Kyu; Jung, Han-Sung; Park, Hwangseo; RYU, CHUN JEIH

    2016-01-01

    Previously, we showed that B-cell receptor associated protein 31 (BAP31), an endoplasmic reticulum (ER) membrane chaperone, is also expressed on the cell surface by two monoclonal antibodies (MAbs) 297-D4 and 144-A8. Both MAbs recognize the same linear epitope on the C-terminal domain of BAP31, although they were independently established. Here, flow cytometric analysis showed that 144-A8 had additional binding properties to some cells, as compared to 297-D4. Quantitative antigen binding assa...

  8. Human pregnancy-associated malaria-specific B cells target polymorphic, conformational epitopes in VAR2CSA

    DEFF Research Database (Denmark)

    Barfod, L.; Bernasconi, N. L.; Dahlback, M.

    2007-01-01

    to evaluate B-cell epitope diversity among parasite isolates, and identified the binding site of one monoclonal antibody using a chimeric DBL3-X construct. Our findings show that there is a high-frequency memory response to VSA(PAM), indicating that VAR2CSA is a primary target of naturally acquired PAM......-molecular-weight (> 200 kDa) proteins, while seven reacted with either the DBL3-X or the DBL5-epsilon domains of VAR2CSA expressed either as Baculovirus constructs or on the surface of transfected Jurkat cells. We used a panel of recombinant antigens representing DBL3-X domains from P. falciparum field isolates...

  9. Revival of the identification of cytotoxic T-lymphocyte epitopes for immunological diagnosis, therapy and vaccine development.

    Science.gov (United States)

    Liu, Jun; Zhang, Shihong; Tan, Shuguang; Zheng, Beiwen; Gao, George F

    2011-03-01

    Immunogenic T-cell epitopes have a central role in the cellular immunity against pathogens and tumors. However, in the early stage of cellular immunity studies, it was complicated and time-consuming to identify and characterize T-cell epitopes. Currently, the epitope screening is experiencing renewed enthusiasm due to advances in novel techniques and theories. Moreover, the application of T-cell epitope-based diagnoses for tuberculosis and new data on epitope-based vaccine development have also revived the field. There is a growing knowledge on the emphasis of epitope-stimulated T-cell immune responses in the elimination of pathogens and tumors. In this review, we outline the significance of the identification and characterization of T-cell epitopes. We also summarize the methods and strategies for epitope definition and, more importantly, address the relevance of cytotoxic T-lymphocyte epitopes to clinical diagnoses, therapy and vaccine development.

  10. Reliable B cell epitope predictions: impacts of method development and improved benchmarking

    DEFF Research Database (Denmark)

    Kringelum, Jens Vindahl; Lundegaard, Claus; Lund, Ole

    2012-01-01

    The interaction between antibodies and antigens is one of the most important immune system mechanisms for clearing infectious organisms from the host. Antibodies bind to antigens at sites referred to as B-cell epitopes. Identification of the exact location of B-cell epitopes is essential in several...... of B-cell epitopes has been moderate. Several issues regarding the evaluation data sets may however have led to the performance values being underestimated: Rarely, all potential epitopes have been mapped on an antigen, and antibodies are generally raised against the antigen in a given biological...... evaluation data set improved from 0.712 to 0.727. Our results thus demonstrate that given proper benchmark definitions, B-cell epitope prediction methods achieve highly significant predictive performances suggesting these tools to be a powerful asset in rational epitope discovery. The updated version...

  11. Prediction and identification of T cell epitopes in the H5N1 influenza virus nucleoprotein in chicken.

    Directory of Open Access Journals (Sweden)

    Yanxia Hou

    Full Text Available T cell epitopes can be used for the accurate monitoring of avian influenza virus (AIV immune responses and the rational design of vaccines. No T cell epitopes have been previously identified in the H5N1 AIV virus nucleoprotein (NP in chickens. For the first time, this study used homology modelling techniques to construct three-dimensional structures of the peptide-binding domains of chicken MHC class Ι molecules for four commonly encountered unique haplotypes, i.e., B4, B12, B15, and B19. H5N1 AIV NP was computationally parsed into octapeptides or nonapeptides according to the peptide-binding motifs of MHC class I molecules of the B4, B12, B15 and B19 haplotypes. Seventy-five peptide sequences were modelled and their MHC class I molecule-binding abilities were analysed by molecular docking. Twenty-five peptides (Ten for B4, six for B12, two for B15, and seven for B19 were predicted to be potential T cell epitopes in chicken. Nine of these peptides and one unrelated peptide were manually synthesized and their T cell responses were tested in vitro. Spleen lymphocytes were collected from SPF chickens that had been immunised with a NP-expression plasmid, pCAGGS-NP, and they were stimulated using the synthesized peptides. The secretion of chicken IFN-γ and the proliferation of CD8(+ T cells were tested using an ELISA kit and flow cytometry, respectively. The significant secretion of chicken IFN-γ and proliferation of CD8(+ T lymphocytes increased by 13.7% and 11.9% were monitored in cells stimulated with peptides NP(89-97 and NP(198-206, respectively. The results indicate that peptides NP(89-97 (PKKTGGPIY and NP(198-206 (KRGINDRNF are NP T cell epitopes in chicken of certain haplotypes. The method used in this investigation is applicable to predicting T cell epitopes for other antigens in chicken, while this study also extends our understanding of the mechanisms of the immune response to AIV in chicken.

  12. Synthetic B-Cell Epitopes Eliciting Cross-Neutralizing Antibodies: Strategies for Future Dengue Vaccine

    OpenAIRE

    Babu Ramanathan; Chit Laa Poh; Kristin Kirk; William John Hannan McBride; John Aaskov; Lara Grollo

    2016-01-01

    Dengue virus (DENV) is a major public health threat worldwide. A key element in protection from dengue fever is the neutralising antibody response. Anti-dengue IgG purified from DENV-2 infected human sera showed reactivity against several peptides when evaluated by ELISA and epitope extraction techniques. A multi-step computational approach predicted six antigenic regions within the E protein of DENV-2 that concur with the 6 epitopes identified by the combined ELISA and epitope extraction app...

  13. Vaccine Focusing to Cross-Subtype HIV-1 gp120 Variable Loop Epitopes

    OpenAIRE

    Cardozo, Timothy; Wang, Shixia; Jiang, Xunqing; Kong, Xiang-Peng; Hioe, Catarina; Krachmarov, Chavdar

    2014-01-01

    We designed synthetic, epitope-focused immunogens that preferentially display individual neutralization epitopes targeted by cross-subtype anti-HIV V3 loop neutralizing monoclonal antibodies (mAbs). Vaccination of rabbits with these immunogens resulted in the elicitation of distinct polyclonal serum Abs that exhibit cross-subtype neutralization specificities mimicking the mAbs that guided the design. Our results prove the principle that a predictable range of epitope-specific polyclonal cross...

  14. Inhibition of serine-peptidase activity enhances the generation of a survivin-derived HLA-A2-presented CTL epitope in colon-carcinoma cells.

    Science.gov (United States)

    Preta, G; Marescotti, D; Fortini, C; Carcoforo, P; Castelli, C; Masucci, M; Gavioli, R

    2008-12-01

    Cytotoxic T lymphocytes eliminate tumor cells expressing antigenic peptides in the context of MHC-I molecules. Peptides are generated during protein degradation by the proteasome and resulting products, surviving cytosolic amino-peptidases activity, may be presented by MHC-I molecules. The MHC-I processing pathway is altered in a large number of malignancies and modulation of antigen generation is one strategy employed by cells to evade immune control. In this study we analyzed the generation and presentation of a survivin-derived CTL epitope in HLA-A2-positive colon-carcinoma cells. Although all cell lines expressed the anti-apoptotic protein survivin, some tumors were poorly recognized by ELTLGEFLKL (ELT)-specific CTL cultures. The expression of MHC-I or TAP molecules was similar in all cell lines suggesting that tumors not recognized by CTLs may present defects in the generation of the ELT-epitope which could be due either to lack of generation or to subsequent degradation of the epitope. The cells were analyzed for the expression and the activity of extra-proteasomal peptidases. A significant overexpression and higher activity of TPPII was observed in colon-carcinoma cells which are not killed by ELT-specific CTLs, suggesting a possible role of TPPII in the degradation of the ELT-epitope. To confirm the role of TPPII in the degradation of the ELT-peptide, we showed that treatment of colon-carcinoma cells with a TPPII inhibitor resulted in a dose-dependent increased sensitivity to ELT-specific CTLs. These results suggest that TPPII is involved in degradation of the ELT-peptide, and its overexpression may contribute to the immune escape of colon-carcinoma cells.

  15. Characterization of a linear epitope on Chlamydia trachomatis serovar L2 DnaK-like protein

    DEFF Research Database (Denmark)

    Ozkokmen, D; Birkelund, Svend; Christiansen, Gunna

    1994-01-01

    A cytoplasmic 75-kDa immunogen from Chlamydia trachomatis serovar L2 has previously been characterized as being similar to the Escherichia coli heat shock protein DnaK. We have localized a linear epitope for one monoclonal antibody specific for C. trachomatis DnaK. By use of a recombinant DNA...... technique, the epitope was limited to 14 amino acids. With synthetic peptides, the epitope was further limited to eight amino acids. Six of these amino acids are conserved in bovine HSP70, which has a known three-dimensional structure. The amino acid sequence homologous to the epitope is located in a linear...

  16. Epitope-dependent functional effects of celiac disease autoantibodies on transglutaminase 2

    DEFF Research Database (Denmark)

    Hnida, Kathrin; Stamnaes, Jorunn; du Pré, M Fleur

    2016-01-01

    Transglutaminase 2 (TG2) is a Ca(2+)-dependent cross-linking enzyme involved in the pathogenesis of CD. We have previously characterized a panel of anti-TG2 mAbs generated from gut plasma cells of celiac patients and identified four epitopes (epitopes 1-4) located in the N-terminal part of TG2...... of epitope 1-targeting B cells to keep TG2 active and protected from oxidation might explain why generation of epitope 1-targeting plasma cells seems to be favored in celiac patients....

  17. Immunogenicity of multiple antigen peptides containing Plasmodium vivax CS epitopes in BALB/c mice

    Directory of Open Access Journals (Sweden)

    Myriam A. Herrera

    1994-01-01

    Full Text Available Multiple antigen peptide systems (MAPs allow the incorporation of various epitopes in to a single synthetic peptide immunogen. We have characterized the immune response of BALB/c mice to a series of MAPs assembled with different B and T cell epitopes derived from the Plasmodium vivax circumsporozoite (CS protein. A B-cell epitope from the central repeat domain and two T-cell epitopes from the amino and carboxyl flanking regions were used to assembled eight different MAPs. An additional universal T cell epitope (ptt-30 from tetanus toxin protein was included. Immunogenicity in terms of antibody responses and in vitro T lymphocyte proliferation was evaluated. MAPs containing B and T cell epitopes induced high titers of anti-peptides antibodies, which recognized the native protein on sporozoites as determined by IFAT. The antibody specificity was also determined by a competitive inhibition assay with different MAPs. A MAP containing the B cell epitope (p11 and the universal epitope ptt-30 together with another composed of p11 and the promiscuous T cell epitope (p25 proved to be the most immunogenic. The strong antibody response and specificity for the cognate protein indicates that further studies designed to assess the potential of these proteins as human malaria vaccine candidates are warranted.

  18. Influenza A HA's conserved epitopes and broadly neutralizing antibodies: a prediction method.

    Science.gov (United States)

    Ren, Jing; Ellis, John; Li, Jinyan

    2014-10-01

    A conserved epitope is an epitope retained by multiple strains of influenza as the key target of a broadly neutralizing antibody. Identification of conserved epitopes is of strong interest to help design broad-spectrum vaccines against influenza. Conservation score measures the evolutionary conservation of an amino acid position in a protein based on the phylogenetic relationships observed amongst homologous sequences. Here, Average Amino Acid Conservation Score (AAACS) is proposed as a method to identify HA's conserved epitopes. Our analysis shows that there is a clear distinction between conserved epitopes and nonconserved epitopes in terms of AAACS. This method also provides an excellent classification performance on an independent dataset. In contrast, alignment-based comparison methods do not work well for this problem, because conserved epitopes to the same broadly neutralizing antibody are usually not identical or similar. Location-based methods are not successful either, because conserved epitopes are located at both the less-conserved globular head (HA1) and the more-conserved stem (HA2). As a case study, two conserved epitopes on HA are predicted for the influenza A virus H7N9: One should match the broadly neutralizing antibodies CR9114 or FI6v3, while the other is new and requires validation by wet-lab experiments.

  19. EpiJen: a server for multistep T cell epitope prediction

    Directory of Open Access Journals (Sweden)

    Guan Pingping

    2006-03-01

    Full Text Available Abstract Background The main processing pathway for MHC class I ligands involves degradation of proteins by the proteasome, followed by transport of products by the transporter associated with antigen processing (TAP to the endoplasmic reticulum (ER, where peptides are bound by MHC class I molecules, and then presented on the cell surface by MHCs. The whole process is modeled here using an integrated approach, which we call EpiJen. EpiJen is based on quantitative matrices, derived by the additive method, and applied successively to select epitopes. EpiJen is available free online. Results To identify epitopes, a source protein is passed through four steps: proteasome cleavage, TAP transport, MHC binding and epitope selection. At each stage, different proportions of non-epitopes are eliminated. The final set of peptides represents no more than 5% of the whole protein sequence and will contain 85% of the true epitopes, as indicated by external validation. Compared to other integrated methods (NetCTL, WAPP and SMM, EpiJen performs best, predicting 61 of the 99 HIV epitopes used in this study. Conclusion EpiJen is a reliable multi-step algorithm for T cell epitope prediction, which belongs to the next generation of in silico T cell epitope identification methods. These methods aim to reduce subsequent experimental work by improving the success rate of epitope prediction.

  20. Optimization and immune recognition of multiple novel conserved HLA-A2, human immunodeficiency virus type 1-specific CTL epitopes

    DEFF Research Database (Denmark)

    Corbet, Sylvie; Nielsen, Henrik Vedel; Vinner, Lasse

    2003-01-01

    and more conserved. Such epitope peptides were anchor-optimized to improve immunogenicity and further increase the number of potential vaccine epitopes. About 67 % of anchor-optimized vaccine epitopes induced immune responses against the corresponding non-immunogenic naturally occurring epitopes....... This study demonstrates the potency of ANNs for identifying putative virus CTL epitopes, and the new HIV-1 CTL epitopes identified should have significant implications for HIV-1 vaccine development. As a novel vaccine approach, it is proposed to increase the coverage of HIV variants by including multiple...

  1. Immune Potential of a Novel Multiple-epitope Vaccine to FMDV Type Asia 1 in Guinea Pigs and Sheep

    Institute of Scientific and Technical Information of China (English)

    Jun-jun Shao; Jing-feng Wang; Hui-yun Chang; Ji-xing Liu

    2011-01-01

    To develop a safe and efficient recombinant subunit vaccine to foot-and-mouth disease virus(FMDV)type Asia 1 in sheep,a tandem repeated multiple-epitope gene consisting of residues 137-160 and 197-211 of the VP1 gene of FMDV was designed and artificially synthesized.The biologically functional molecule,the ovine IgG heavy constant region(oIgG)as a protein carrier was introduced for design of the multiple-epitope recombinant vaccine and recombinant expression plasmids pET-30a-RE and pET-30a-RE-oIgG were successfully constructed.The recombinant proteins,RE and RE-oIgG,were expressed as a formation of inclusion bodies in E.coli.The immune potential of this vaccine regime in guinea pigs and sheep was evaluated.The results showed that IgG could significantly enhance the immune potential of antigenic epitopes.The recombinant protein RE-oIgG could not only elicit the high levels of neutralizing antibodies and lymphocytes proliferation responses in the vaccinated guinea pigs,but confer complete protection in guinea pigs against virus challenge.Although the recombinant protein RE could not confer protection in the vaccinated animals,it could delay the appearance of the clinical signs and reduce the severity of disease.Inspiringly,the titers of anti-FMDV neutralizing antibodies elicited in sheep vaccinated with RE-oIgG was significantly higher than that for the RE vaccination.Therefore,we speculated that this vaccine formulation may be a promising strategy for designing a novel vaccine against FMDV in the future.

  2. Evaluation of a chimeric multi-epitope-based DNA vaccine against subgroup J avian leukosis virus in chickens.

    Science.gov (United States)

    Xu, Qingqing; Cui, Ning; Ma, Xingjiang; Wang, Fangkun; Li, Hongmei; Shen, Zhiqiang; Zhao, Xiaomin

    2016-07-19

    The prokaryotic expressed recombinant chimeric multi-epitope protein X (rCMEPX) had been evaluated with good immunogenicity and protective efficacy against subgroup J avian leukosis virus (ALV-J) in our previous study. In the present research, we cloned the chimeric multi-epitope gene X into the eukaryotic expression vector pVAX1 to evaluate its potency as a DNA vaccine. The purified recombinant gp85 protein and rCMEPX were used as positive controls and a DNA prime-protein boost strategy was also studied. Six experimental groups of 7-day-old chickens (20 per group) were immunized intramuscularly three times at 2weeks interval with PBS, gp85, rCMEPX, pVAX1, pVAX-X and pVAX-X+rCMEPX respectively. The antibody titers and cellular immune responses were assayed after immunization. The efficacy of immunoprotection against the challenge of ALV-J NX0101 strain was also examined. The results showed that the DNA vaccine could elicit both neutralizing antibodies and cellular responses. Immune-challenge experiments showed good protection efficacy against ALV-J infection. Particularly, the regimen involving one priming pVAX-X and twice recombinant rCMEPX boosting, induced the highest antibody titers in all immunized groups. Our results suggest that the constructed chimeric multi-epitope DNA has potential for a candidate vaccine against ALV-J when used in proper prime-boost combinations. The data presented here may provide an alternative strategy for vaccine design in chicken ALV-J prevention.

  3. Three novel NY-ESO-1 epitopes bound to DRB1*0803, DQB1*0401 and DRB1*0901 recognized by CD4 T cells from CHP-NY-ESO-1-vaccinated patients.

    Science.gov (United States)

    Mizote, Yu; Taniguchi, Taku; Tanaka, Kei; Isobe, Midori; Wada, Hisashi; Saika, Takashi; Kita, Shoichi; Koide, Yukari; Uenaka, Akiko; Nakayama, Eiichi

    2010-07-19

    Three novel NY-ESO-1 CD4 T cell epitopes were identified using PBMC obtained from patients who were vaccinated with a complex of cholesterol-bearing hydrophobized pullulan (CHP) and NY-ESO-1 protein (CHP-NY-ESO-1). The restriction molecules were determined by antibody blocking and using various EBV-B cells with different HLA alleles as APC to present peptides to CD4 T cells. The minimal epitope peptides were determined using various N- and C-termini truncated peptides deduced from 18-mer overlapping peptides originally identified for recognition. Those epitopes were DRB1*0901-restricted NY-ESO-1 87-100, DQB1*0401-restricted NY-ESO-1 95-107 and DRB1*0803-restricted NY-ESO-1 124-134. CD4 T cells used to determine those epitope peptides recognized EBV-B cells or DC that were treated with recombinant NY-ESO-1 protein or NY-ESO-1-expressing tumor cell lysate, suggesting that the epitope peptides are naturally processed. These CD4 T cells showed a cytokine profile with Th1 characteristics. Furthermore, NY-ESO-1 87-100 peptide/HLA-DRB1*0901 tetramer staining was observed. Multiple Th1-type CD4 T cell responses are beneficial for inducing effective anti-tumor responses after NY-ESO-1 protein vaccination.

  4. Plasmodium vivax Promiscuous T-Helper Epitopes Defined and Evaluated as Linear Peptide Chimera Immunogens

    Science.gov (United States)

    Caro-Aguilar, Ivette; Rodríguez, Alexandra; Calvo-Calle, J. Mauricio; Guzmán, Fanny; De la Vega, Patricia; Elkin Patarroyo, Manuel; Galinski, Mary R.; Moreno, Alberto

    2002-01-01

    Clinical trials of malaria vaccines have confirmed that parasite-derived T-cell epitopes are required to elicit consistent and long-lasting immune responses. We report here the identification and functional characterization of six T-cell epitopes that are present in the merozoite surface protein-1 of Plasmodium vivax (PvMSP-1) and bind promiscuously to four different HLA-DRB1∗ alleles. Each of these peptides induced lymphoproliferative responses in cells from individuals with previous P. vivax infections. Furthermore, linear-peptide chimeras containing the promiscuous PvMSP-1 T-cell epitopes, synthesized in tandem with the Plasmodium falciparum immunodominant circumsporozoite protein (CSP) B-cell epitope, induced high specific antibody titers, cytokine production, long-lasting immune responses, and immunoglobulin G isotype class switching in BALB/c mice. A linear-peptide chimera containing an allele-restricted P. falciparum T-cell epitope with the CSP B-cell epitope was not effective. Two out of the six promiscuous T-cell epitopes exhibiting the highest anti-peptide response also contain B-cell epitopes. Antisera generated against these B-cell epitopes recognize P. vivax merozoites in immunofluorescence assays. Importantly, the anti-peptide antibodies generated to the CSP B-cell epitope inhibited the invasion of P. falciparum sporozoites into human hepatocytes. These data and the simplicity of design of the chimeric constructs highlight the potential of multimeric, multistage, and multispecies linear-peptide chimeras containing parasite promiscuous T-cell epitopes for malaria vaccine development. PMID:12065487

  5. A strategy for eliciting antibodies against cryptic, conserved, conformationally dependent epitopes of HIV envelope glycoprotein.

    Directory of Open Access Journals (Sweden)

    Hanna C Kelker

    Full Text Available BACKGROUND: Novel strategies are needed for the elicitation of broadly neutralizing antibodies to the HIV envelope glycoprotein, gp120. Experimental evidence suggests that combinations of antibodies that are broadly neutralizing in vitro may protect against challenge with HIV in nonhuman primates, and a small number of these antibodies have been selected by repertoire sampling of B cells and by the fractionation of antiserum from some patients with prolonged disease. Yet no additional strategies for identifying conserved epitopes, eliciting antibodies to these epitopes, and determining whether these epitopes are accessible to antibodies have been successful to date. The defining of additional conserved, accessible epitopes against which one can elicit antibodies will increase the probability that some may be the targets of broadly neutralizing antibodies. METHODOLOGY/PRINCIPAL FINDINGS: We postulate that additional cryptic epitopes of gp120 are present, against which neutralizing antibodies might be elicited even though these antibodies are not elicited by gp120, and that many of these epitopes may be accessible to antibodies should they be formed. We demonstrate a strategy for eliciting antibodies in mice against selected cryptic, conformationally dependent conserved epitopes of gp120 by immunizing with multiple identical copies of covalently linked peptides (MCPs. This has been achieved with MCPs representing 3 different domains of gp120. We show that some cryptic epitopes on gp120 are accessible to the elicited antibodies, and some epitopes in the CD4 binding region are not accessible. The antibodies bind to gp120 with relatively high affinity, and bind to oligomeric gp120 on the surface of infected cells. CONCLUSIONS/SIGNIFICANCE: Immunization with MCPs comprised of selected peptides of HIV gp120 is able to elicit antibodies against conserved, conformationally dependent epitopes of gp120 that are not immunogenic when presented as gp120. Some

  6. IMMUNOCAT—A Data Management System for Epitope Mapping Studies

    Directory of Open Access Journals (Sweden)

    Jo L. Chung

    2010-01-01

    Full Text Available To enable rationale vaccine design, studies of molecular and cellular mechanisms of immune recognition need to be linked with clinical studies in humans. A major challenge in conducting such translational research studies lies in the management and integration of large amounts and various types of data collected from multiple sources. For this purpose, we have established “IMMUNOCAT”, an interactive data management system for the epitope discovery research projects conducted by our group. The system provides functions to store, query, and analyze clinical and experimental data, enabling efficient, systematic, and integrative data management. We demonstrate how IMMUNOCAT is utilized in a large-scale research contract that aims to identify epitopes in common allergens recognized by T cells from human donors, in order to facilitate the rational design of allergy vaccines. At clinical sites, demographic information and disease history of each enrolled donor are captured, followed by results of an allergen skin test and blood draw. At the laboratory site, T cells derived from blood samples are tested for reactivity against a panel of peptides derived from common human allergens. IMMUNOCAT stores results from these T cell assays along with MHC:peptide binding data, results from RAST tests for antibody titers in donor serum, and the respective donor HLA typing results. Through this system, we are able to perform queries and integrated analyses of the various types of data. This provides a case study for the use of bioinformatics and information management techniques to track and analyze data produced in a translational research study aimed at epitope identification.

  7. IMMUNOCAT-a data management system for epitope mapping studies.

    Science.gov (United States)

    Chung, Jo L; Sun, Jian; Sidney, John; Sette, Alessandro; Peters, Bjoern

    2010-01-01

    To enable rationale vaccine design, studies of molecular and cellular mechanisms of immune recognition need to be linked with clinical studies in humans. A major challenge in conducting such translational research studies lies in the management and integration of large amounts and various types of data collected from multiple sources. For this purpose, we have established "IMMUNOCAT", an interactive data management system for the epitope discovery research projects conducted by our group. The system provides functions to store, query, and analyze clinical and experimental data, enabling efficient, systematic, and integrative data management. We demonstrate how IMMUNOCAT is utilized in a large-scale research contract that aims to identify epitopes in common allergens recognized by T cells from human donors, in order to facilitate the rational design of allergy vaccines. At clinical sites, demographic information and disease history of each enrolled donor are captured, followed by results of an allergen skin test and blood draw. At the laboratory site, T cells derived from blood samples are tested for reactivity against a panel of peptides derived from common human allergens. IMMUNOCAT stores results from these T cell assays along with MHC:peptide binding data, results from RAST tests for antibody titers in donor serum, and the respective donor HLA typing results. Through this system, we are able to perform queries and integrated analyses of the various types of data. This provides a case study for the use of bioinformatics and information management techniques to track and analyze data produced in a translational research study aimed at epitope identification.

  8. Some epitopes conservation in non structural 3 protein dengue virus serotype 4

    Directory of Open Access Journals (Sweden)

    Tegar A. P. Siregar

    2016-03-01

    Full Text Available AbstrakLatar belakang: Protein Non Struktural 3 (NS3 virus dengue menginduksi respon antibodi netralisasidan respon sel T CD4+ dan CD8+, serta berperan dalam replikasi virus. Protein NS3 memiliki epitopepitopsel T dan B yang terdapat perbedaan kelestarian pada berbagai strain virus dengue serotipe 4(DENV-4. Penelitian ini bertujuan untuk mengetahui kelestarian epitop sel T dan B pada protein NS3DENV-4 strain-strain dunia dan keempat serotipe virus dengue strain Indonesia.Metode: Penelitian ini dilakukan di Departemen Mikrobiologi Fakultas Kedokteran UI sejak Juni 2013 - April2014. Sekuens asam amino NS3 DENV-4 strain 081 didapatkan setelah produk PCR gen NS3 DENV-4 081disekuensing. Epitop-epitop sel T dan sel B protein NS3 DENV-4 081 dianalisis dan dibandingkan dengansekuens asam amino protein NS3 dari 124 strain DENV-4 di dunia dan keempat serotipe DENV strain Indonesia.Strain-strain dunia merupakan strain yang ada di benua Amerika (Venezuela, Colombia, dll dan Asia (Cina,Singapura, dll. Referensi posisi epitop sel T dan B protein NS3 diperoleh dari laporan penelitian terdahulu.Hasil: Delapan epitop sel T dan 2 epitop sel B dari protein NS3 DENV-4 081 ternyata identik dan lestaripada protein NS3 dari 124 strain DENV-4 dunia. Epitop sel B di posisi asam amino 537-544 pada proteinNS3 DENV-4 081 ternyata identik dan lestari dengan epitop sel B protein NS3 dari keempat serotipeDENV strain Indonesia.Kesimpulan: Kelestarian yang luas dari epitop sel T dan B pada hampir seluruh strain DENV-4 dunia danserotipe-serotipe DENV strain Indonesia. (Health Science Journal of Indonesia 2015;6:126-31Kata kunci: virus dengue, protein NS3, epitop sel T, epitop sel B AbstractBackground: Non Structural 3 (NS3 protein of dengue virus (DENV is known to induce antibody, CD4+and CD8+ T cell responses, and playing role in viral replication. NS3 protein has T and B cell epitopes,which has conservation difference between DENV-4 strains. This study aimed to identify

  9. Oral immunogenicity of tomato-derived sDPT polypeptide containing Corynebacterium diphtheriae, Bordetella pertussis and Clostridium tetani exotoxin epitopes.

    Science.gov (United States)

    Soria-Guerra, Ruth E; Rosales-Mendoza, Sergio; Moreno-Fierros, Leticia; López-Revilla, Rubén; Alpuche-Solís, Angel G

    2011-03-01

    DPT vaccine, designed to immunize against diphtheria, pertussis, and tetanus, has been shown to be effective in humans. Nevertheless, dissatisfaction with the whole-cell preparations is due to the reactogenicity, which has to lead to the development of new safer formulations. Previously, we described the expression in tomato of a plant-optimized synthetic gene encoding the recombinant polypeptide sDPT, containing mainly immunoprotective epitopes of the diphtheria, pertussis and tetanus exotoxins and two adjuvants. In this study, we examined whether the ingestion of tomato-derived sDPT protein induces specific antibodies in mice after three weekly doses scheme. A positive group immunized with DPT toxoids was included. Specific antibody levels were assessed in serum, gut and lung. Sera tested for IgG antibody response to pertussis, tetanus and diphtheria toxin showed responses to the foreign antigens; interestingly, the response to diphtheria epitope was similar to those observed in the positive group. We found higher IgG1 than IgG2a responses in serum. A modest IgG response was observed in the tracheopulmonary fluid. High response of IgA against tetanus toxin was evident in gut, which was statistically comparable to that obtained in the positive group. The levels of response in these groups were higher than those in mice that received wild-type tomato. These findings support the concept of using transgenic tomatoes expressing sDPT polypeptide as model for edible vaccine against diphtheria, pertussis, and tetanus.

  10. FMC46, a cell protrusion-associated leukocyte adhesion molecule-1 epitope on human lymphocytes and thymocytes.

    Science.gov (United States)

    Pilarski, L M; Turley, E A; Shaw, A R; Gallatin, W M; Laderoute, M P; Gillitzer, R; Beckman, I G; Zola, H

    1991-07-01

    In this report, we describe a 76-kDa glycoprotein recognized by mAb FMC46 that, by virtue of its concentration on cell protrusions involved in motility, may be important in lymphoid cell locomotion. FMC46 detects an epitope of the leukocyte adhesion molecule-1 (LAM-1), a member of the selecting family (LAM-1, Endothelial Leukocyte Adhesion Molecular-1 (ELAM-1), and Granule Membrane Protein-140 (GMP-140), that is expressed on LAM-1-transfected cell lines, is a glycosylation epitope based on its loss after culture in tunicamycin, and is closely related to the LAM-1.2 epitope. FMC46 is expressed at high density on the majority of CD45RA+ and CD45RO+ peripheral blood T cells (60 to 70%) and on a subset of thymocytes that includes the multinegative CD3- CD4- CD8- progenitor cells (100% FMC46hi) and the CD45R0- presumptive thymic generative lineage (70% FMC46hi). It appears at reduced density and frequency on CD45RA- thymocytes (50% FMC46lo), comprised mainly of death-committed thymocytes. Among thymic subsets defined by expression of CD4 and/or CD8, FMC46 is expressed at high density predominantly on a subset of single-positive cells and not on double-positive cells. These results suggest a fundamental role for LAM-1 in thymic development, with a high density preferentially expressed on cells involved in thymic generative processes and a low density on cells progressing to intrathymic death. A major subset of peripheral blood B cells and thymic B cells also express FMC46. Immunohistochemistry on frozen sections indicated strong staining in splenic follicles and around blood vessels, staining of the thymic medulla and subcapsular areas, and staining of the mantle zone of germinal centers of the lymph node. FMC46+ lymphocytes accumulated along high endothelial venules in the lymph node. On locomoting multinegative thymocytes, FMC46 is concentrated on the leading tip of extended processes, on pseudopods, and on ruffles, unlike the distribution of either CD44 or TQ1 (LAM 1

  11. Conflicting selective forces affect T cell receptor contacts in an immunodominant human immunodeficiency virus epitope

    DEFF Research Database (Denmark)

    Iversen, Astrid K N; Stewart-Jones, Guillaume; Learn, Gerald H

    2006-01-01

    Cytotoxic T lymphocytes (CTLs) are critical for the control of human immunodeficiency virus, but containment of virus replication can be undermined by mutations in CTL epitopes that lead to virus escape. We analyzed the evolution in vivo of an immunodominant, HLA-A2-restricted CTL epitope and fou...

  12. Collection of phage-peptide probes for HIV-1 immunodominant loop-epitope.

    Science.gov (United States)

    Palacios-Rodríguez, Yadira; Gazarian, Tatiana; Rowley, Merrill; Majluf-Cruz, Abraham; Gazarian, Karlen

    2007-02-01

    Early diagnosis and prevention of human immunodeficiency virus type-1 (HIV-1) infection, which remains a serious public health threat, is inhibited by the lack of reagents that elicit antiviral responses in the immune system. To create mimotopes (peptide models of epitopes) of the most immunodominant epitope, CSGKLIC, that occurs as a loop on the envelope gp41 glycoprotein and is a key participant in infection, we used phage-display technology involving biopanning of large random libraries with IgG of HIV-1-infected patients. Under the conditions used, library screening with IgG from patient serum was directed to the CSGKLIC epitope. Three rounds of selection converted a 12 mer library of 10(9) sequences into a population in which up to 79% of phage bore a family of CxxKxxC sequences ("x" designates a non-epitope amino acid). Twenty-one phage clones displaying the most frequently selected peptides were obtained and were shown to display the principal structural (sequence and conformational), antigenic and immunogenic features of the HIV-1 immunodominant loop-epitope. Notably, when the mixture of the phage mimotopes was injected into mice, it induced 2- to 3-fold higher titers of antibody to the HIV-1 epitope than could be induced from individual mimotopes. The described approach could be applicable for accurately reproducing HIV-1 epitope structural and immunological patterns by generation of specialized viral epitope libraries for use in diagnosis and therapy.

  13. Towards a consensus on datasets and evaluation metrics for developing B-cell epitope prediction tools

    DEFF Research Database (Denmark)

    Greenbaum, Jason A.; Andersen, Pernille; Blythe, Martin

    2007-01-01

    A B-cell epitope is the three-dimensional structure within an antigen that can be bound to the variable region of an antibody. The prediction of B-cell epitopes is highly desirable for various immunological applications, but has presented a set of unique challenges to the bioinformatics and immun...

  14. B-Cell Receptor Epitope Recognition Correlates With the Clinical Course of Chronic Lymphocytic Leukemia

    NARCIS (Netherlands)

    Binder, Mascha; Mueller, Fabian; Jackst, Antje; Lechenne, Barbara; Pantic, Milena; Bacher, Ulrike; Eulenburg, Christine Zu; Veelken, Hendrik; Mertelsmann, Roland; Pasqualini, Renata; Arap, Wadih; Trepel, Martin

    2011-01-01

    BACKGROUND: B-cell receptors (BCRs) and their recognition of specific epitopes may play a pivotal role in the development and progression of chronic lymphocytic leukemia (CLL). In this study, the authors set up a model system to explore epitope reactivity and its clinical relevance in CLL. METHODS:

  15. Computational prediction of neutralization epitopes targeted by human anti-V3 HIV monoclonal antibodies.

    Directory of Open Access Journals (Sweden)

    Evgeny Shmelkov

    Full Text Available The extreme diversity of HIV-1 strains presents a formidable challenge for HIV-1 vaccine design. Although antibodies (Abs can neutralize HIV-1 and potentially protect against infection, antibodies that target the immunogenic viral surface protein gp120 have widely variable and poorly predictable cross-strain reactivity. Here, we developed a novel computational approach, the Method of Dynamic Epitopes, for identification of neutralization epitopes targeted by anti-HIV-1 monoclonal antibodies (mAbs. Our data demonstrate that this approach, based purely on calculated energetics and 3D structural information, accurately predicts the presence of neutralization epitopes targeted by V3-specific mAbs 2219 and 447-52D in any HIV-1 strain. The method was used to calculate the range of conservation of these specific epitopes across all circulating HIV-1 viruses. Accurately identifying an Ab-targeted neutralization epitope in a virus by computational means enables easy prediction of the breadth of reactivity of specific mAbs across the diversity of thousands of different circulating HIV-1 variants and facilitates rational design and selection of immunogens mimicking specific mAb-targeted epitopes in a multivalent HIV-1 vaccine. The defined epitopes can also be used for the purpose of epitope-specific analyses of breakthrough sequences recorded in vaccine clinical trials. Thus, our study is a prototype for a valuable tool for rational HIV-1 vaccine design.

  16. Identification and fine mapping of a linear B cell epitope of human vimentin

    DEFF Research Database (Denmark)

    Dam, Catharina Essendrup; Houen, Gunnar; Hansen, Paul R.

    2014-01-01

    Knowledge about antibody-antigen interactions is important for the understanding of the immune system mechanisms and for supporting development of drugs and biomarkers. A tool for identification of these antigenic epitopes of specific antibodies is epitope mapping. In this study, a modified enzym...

  17. IgE epitopes of intact and digested Ara h 1

    DEFF Research Database (Denmark)

    Bøgh, Katrine Lindholm; Nielsen, H.; Madsen, Charlotte Bernhard;

    2012-01-01

    epitopes have been suggested to be of great importance. ObjectiveThe aim of this study was to identify IgE specific epitopes of intact and digested Ara h 1, and to compare epitope patterns between humans and rats. MethodsSera from five peanut allergic patients and five Brown Norway rats were used...... to identify intact and digested Ara h 1-specific IgE epitopes by competitive immunoscreening of a phage-displayed random hepta-mer peptide library using polyclonal IgE from the individual sera. The resulting peptide sequences were mapped on the surface of a three-dimensional structure of the Ara h 1 molecule...... to mimic epitopes using a computer-based algorithm. ResultsPatients as well as rats were shown to have individual IgE epitope patterns. All epitope mimics were conformational and found to cluster into three different areas of the Ara h 1 molecule. Five epitope motifs were identified by patient IgE, which...

  18. Structural analysis of B-cell epitopes in antibody:protein complexes

    DEFF Research Database (Denmark)

    Kringelum, Jens Vindahl; Nielsen, Morten; Padkjær, Søren Berg;

    2013-01-01

    developed a novel framework for comparing and superimposing B-cell epitopes and applied it on a dataset of 107 non-similar antigen:antibody structures extracted from the PDB database. With the presented framework, we were able to describe the general B-cell epitope as a flat, oblong, oval shaped volume...

  19. Epitope-Specific Suppression of IgG Responses by Passively Administered Specific IgG: Evidence of Epitope Masking

    Science.gov (United States)

    Bergström, Joakim J. E.; Xu, Hui; Heyman, Birgitta

    2017-01-01

    Specific IgG, passively administered together with particulate antigen, can completely prevent induction of antibody responses to this antigen. The ability of IgG to suppress antibody responses to sheep red blood cells (SRBCs) is intact in mice lacking FcγRs, complement factor 1q, C3, or complement receptors 1 and 2, suggesting that Fc-dependent effector functions are not involved. Two of the most widely discussed explanations for the suppressive effect are increased clearance of IgG–antigen complexes and/or that IgG “hides” the antigen from recognition by specific B cells, so-called epitope masking. The majority of data on how IgG induces suppression was obtained through studies of the effects on IgM-secreting single spleen cells during the first week after immunization. Here, we show that IgG also suppresses antigen-specific extrafollicular antibody-secreting cells, germinal center B-cells, long-lived plasma cells, long-term IgG responses, and induction of memory antibody responses. IgG anti-SRBC reduced the amount of SRBC in the spleens of wild-type, but not of FcγR-deficient mice. However, no correlation between suppression and the amount of SRBC in the spleen was observed, suggesting that increased clearance does not explain IgG-mediated suppression. Instead, we found compelling evidence for epitope masking because IgG anti-NP administered with NP-SRBC suppressed the IgG anti-NP, but not the IgG anti-SRBC response. Vice versa, IgG anti-SRBC administered with NP-SRBC, suppressed only the IgG anti-SRBC response. In conclusion, passively transferred IgG suppressed all measured parameters of an antigen-specific antibody/B cell response and an important mechanism of action is likely to be epitope masking.

  20. Neisseria lactamica and Neisseria meningitidis share lipooligosaccharide epitopes but lack common capsular and class 1, 2, and 3 protein epitopes.

    Science.gov (United States)

    Kim, J J; Mandrell, R E; Griffiss, J M

    1989-02-01

    Neisseria lactamica, a common human pharyngeal commensal, contributes to acquired immunity to Neisseria meningitidis. To define the surface antigens shared between these two species, we used monoclonal antibodies (MAbs) to study 35 N. lactamica strains isolated in various parts of the world for cross-reactivity with meningococcal capsules, outer membrane proteins, and lipooligosaccharides (LOS). No N. lactamica strain reacted significantly with MAbs specific for capsular group A, B, C, Y, or W, and we were unable to extract capsular polysaccharide from them. Only 2 of 33 strains reacted weakly with MAbs against class 2 serotype proteins P2b and P2c. None reacted with MAbs specific for meningococcal class 1 protein P1.2 or P1.16 or class 2/3 serotype protein P2a or P15. Most N. lactamica strains (30 of 35) bound one or more of seven LOS-specific MAbs. Two LOS epitopes, defined by MAbs O6B4 and 3F11, that are commonly found on pathogenic Neisseria species were found on 25 of 35 N. lactamica. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting showed that the LOS of N. lactamica are composed of multiple components that are physically and antigenically similar to the LOS of pathogenic Neisseria species. Among four other commensal neisserial species, only Neisseria cinerea shared LOS epitopes defined by MAbs O6B4 and 3F11. Previous studies have shown that pharyngeal colonization with N. lactamica induces bactericidal antibodies against the meningococcus. We postulate that shared N. lactamica and meningococcal LOS epitopes may play an important role in the development of natural immunity to the meningococcus.

  1. Codon optimization of the human papillomavirus E7 oncogene induces a CD8+ T cell response to a cryptic epitope not harbored by wild-type E7.

    Directory of Open Access Journals (Sweden)

    Felix K M Lorenz

    Full Text Available Codon optimization of nucleotide sequences is a widely used method to achieve high levels of transgene expression for basic and clinical research. Until now, immunological side effects have not been described. To trigger T cell responses against human papillomavirus, we incubated T cells with dendritic cells that were pulsed with RNA encoding the codon-optimized E7 oncogene. All T cell receptors isolated from responding T cell clones recognized target cells expressing the codon-optimized E7 gene but not the wild type E7 sequence. Epitope mapping revealed recognition of a cryptic epitope from the +3 alternative reading frame of codon-optimized E7, which is not encoded by the wild type E7 sequence. The introduction of a stop codon into the +3 alternative reading frame protected the transgene product from recognition by T cell receptor gene-modified T cells. This is the first experimental study demonstrating that codon optimization can render a transgene artificially immunogenic through generation of a dominant cryptic epitope. This finding may be of great importance for the clinical field of gene therapy to avoid rejection of gene-corrected cells and for the design of DNA- and RNA-based vaccines, where codon optimization may artificially add a strong immunogenic component to the vaccine.

  2. Identification of a Common Epitope between Enterovirus 71 and Human MED25 Proteins Which May Explain Virus-Associated Neurological Disease

    Directory of Open Access Journals (Sweden)

    Peihu Fan

    2015-03-01

    Full Text Available Enterovirus 71 (EV71 is a major causative pathogen of hand, foot and mouth disease with especially severe neurologic complications, which mainly account for fatalities from this disease. To date, the pathogenesis of EV71 in the central neurons system has remained unclear. Cytokine-mediated immunopathogenesis and nervous tissue damage by virus proliferation are two widely speculated causes of the neurological disease. To further study the pathogenesis, we identified a common epitope (co-epitope between EV71 VP1 and human mediator complex subunit 25 (MED25 highly expressed in brain stem. A monoclonal antibody (2H2 against the co-epitope was prepared, and its interaction with MED25 was examined by ELISA, immunofluorescence assay and Western blot in vitro and by live small animal imaging in vivo. Additionally, 2H2 could bind to both VP1 and MED25 with the affinity constant (Kd of 10−7 M as determined by the ForteBio Octet System. Intravenously injected 2H2 was distributed in brain stem of mice after seven days of EV71 infection. Interestingly, 2H2-like antibodies were detected in the serum of EV71-infected patients. These findings suggest that EV71 infection induces the production of antibodies that can bind to autoantigens expressed in nervous tissue and maybe further trigger autoimmune reactions resulting in neurological disease.

  3. Identification of novel helper epitope peptides of Survivin cancer-associated antigen applicable to developing helper/killer-hybrid epitope long peptide cancer vaccine.

    Science.gov (United States)

    Ohtake, Junya; Ohkuri, Takayuki; Togashi, Yuji; Kitamura, Hidemitsu; Okuno, Kiyotaka; Nishimura, Takashi

    2014-09-01

    We identified novel helper epitope peptides of Survivin cancer antigen, which are presented to both HLA-DRB1*01:01 and DQB1*06:01. The helper epitope also contained three distinct Survivin-killer epitopes presented to HLA-A*02:01 and A*24:02. This 19 amino-acids epitope peptide (SU18) induced weak responses of Survivin-specific CD4(+) and CD8(+) T cells though it contained both helper and killer epitopes. To enhance the vaccine efficacy, we synthesized a long peptide by conjugating SU18 peptide and another DR53-restricted helper epitope peptide (SU22; 12 amino-acids) using glycine-linker. We designated this artificial 40 amino-acids long peptide containing two helper and three killer epitopes as Survivin-helper/killer-hybrid epitope long peptide (Survivin-H/K-HELP). Survivin-H/K-HELP allowed superior activation of IFN-γ-producing CD4(+) Th1 cells and CD8(+) Tc1 cells compared with the mixture of its component peptides (SU18 and SU22) in the presence of OK-432-treated monocyte-derived DC (Mo-DC). Survivin-H/K-HELP-pulsed Mo-DC pretreated with OK-432 also exhibited sustained antigen-presentation capability of stimulating Survivin-specific Th1 cells compared with Mo-DC pulsed with a mixture of SU18 and SU22 short peptides. Moreover, we demonstrated that Survivin-H/K-HELP induced a complete response in a breast cancer patient with the induction of cellular and humoral immune responses. Thus, we believe that an artificially synthesized Survivin-H/K-HELP will become an innovative cancer vaccine.

  4. Further progress on defining highly conserved immunogenic epitopes for a global HIV vaccine

    DEFF Research Database (Denmark)

    De Groot, Anne S; Levitz, Lauren; Ardito, Matthew T;

    2012-01-01

    and that are conserved in sequence and across time may represent the "Achilles' heel" of HIV and would be excellent candidates for vaccine development. In this study, T-cell epitopes were selected using immunoinformatics tools, combining HLA-A3 binding predictions with relative sequence conservation in the context...... of global HIV evolution. Twenty-seven HLA-A3 epitopes were chosen from an analysis performed in 2003 on 10,803 HIV-1 sequences, and additional sequences were selected in 2009 based on an expanded set of 43,822 sequences. These epitopes were tested in vitro for HLA binding and for immunogenicity with PBMCs...... of HIV-infected donors from Providence, Rhode Island. Validation of these HLA-A3 epitopes conserved across time, clades, and geography supports the hypothesis that epitopes such as these would be candidates for inclusion in our globally relevant GAIA HIV vaccine constructs....

  5. 表位疫苗的研究进展%Advance in epitope vaccines

    Institute of Scientific and Technical Information of China (English)

    石晓妮; 窦永喜; 才学鹏

    2011-01-01

    综述了表位疫苗的免疫学理论基础、抗原表位的预测及构建表位疫苗的基本思路和方法,介绍了目前国内外已经研制成功的几种病原表位疫苗,为深入研制其他病原微生物的表位疫苗提供借鉴.%This article reviews the basic immune mechanisms of epitope vaccines and the methods on how to predict antigenic epitopes and design epitope vaccines.Some examples of successful epitope vaccines were included.From them,researchers may be able to get some useful information and help to develop new epitope vaccine.

  6. High throughput functional epitope mapping: revisiting phage display platform to scan target antigen surface.

    Science.gov (United States)

    Rojas, Gertrudis; Tundidor, Yaima; Infante, Yanelys Cabrera

    2014-01-01

    Antibody engineering must be accompanied by mapping strategies focused on identifying the epitope recognized by each antibody to define its unique functional identity. High throughput fine specificity determination remains technically challenging. We review recent experiences aimed at revisiting the oldest and most extended display technology to develop a robust epitope mapping platform, based on the ability to manipulate target-derived molecules (ranging from the whole native antigen to antigen domains and smaller fragments) on filamentous phages. Single, multiple and combinatorial mutagenesis allowed comprehensive scanning of phage-displayed antigen surface that resulted in the identification of clusters of residues contributing to epitope formation. Functional pictures of the epitope(s) were thus delineated in the natural context. Successful mapping of antibodies against interleukin-2, epidermal growth factor and its receptor, and vascular endothelial growth factor showed the versatility of these procedures, which combine the accuracy of site-directed mutagenesis with the high throughput potential of phage display.

  7. Identification of a human immunodominant B-cell epitope within the immunoglobulin A1 protease of Streptococcus pneumoniae

    Directory of Open Access Journals (Sweden)

    Felici Franco

    2007-12-01

    Full Text Available Abstract Background The IgA1 protease of Streptococcus pneumoniae is a proteolytic enzyme that specifically cleaves the hinge regions of human IgA1, which dominates most mucosal surfaces and is the major IgA isotype in serum. This protease is expressed in all of the known pneumococcal strains and plays a major role in pathogen's resistance to the host immune response. The present work was focused at identifying the immunodominant regions of pneumococcal IgA1 protease recognized by the human antibody response. Results An antigenic sequence corresponding to amino acids 420–457 (epiA of the iga gene product was identified by screening a pneumococcal phage display library with patients' sera. The epiA peptide is conserved in all pneumococci and in two out of three S. mitis strains, while it is not present in other oral streptococci so far sequenced. This epitope was specifically recognized by antibodies present in sera from 90% of healthy adults, thus representing an important target of the humoral response to S. pneumoniae and S. mitis infection. Moreover, sera from 68% of children less than 4 years old reacted with the epiA peptide, indicating that the human immune response against streptococcal antigens occurs during childhood. Conclusion The broad and specific recognition of the epiA polypeptide by human sera demonstrate that the pneumococcal IgA1 protease contains an immunodominant B-cell epitope. The use of phage display libraries to identify microbe or disease-specific antigens recognized by human sera is a valuable approach to epitope discovery.

  8. Epitope mapping and identification on a 3D model built for the tartary buckwheat allergic protein TBb

    Institute of Scientific and Technical Information of China (English)

    Ping Li; Xiaodong Cui; Yuying Li; Zhuanhua Wang

    2011-01-01

    Allergic protein TBb, a major allergen in tartary buckwheat, was divided into four epitope-containing fragments and was named Fl, F2, F3, and F4, respectively.Results of immunological assays revealed that F2 had the strongest IgE-binding activity to patient's sera, which indicated that it might contain the linear IgE-binding epitope of TBb.According to the results of sequence analysis and molecular modeling of tartary buckwheat allergen, three mutants of F2 gene (R139A, R141A, and D144A) were reconstructed using site-directed mutagenesis, and each mutant was expressed in Escherichia coli BL21 (DE3).Following purification by Ni2+ affinity chromatography, enzyme-linked immunosorbent assay and dot blot were performed for wild-type F2 and its mutants using sera from buckwheat-allergic patients and a negative control (non-allergic patient).Results showed that mutants R139A and D144A had weaker IgE-binding activity to patient's sera than wild-type F2, implying that Arg139 and Asp144 might be involved in the allergic activity of TBb.However, R141A had the weakest IgE-binding activity,suggesting that Arg141 may be the critical amino acid of TBb.This is the first report on the epitope mapping and identification of TBb.Our findings will contribute to the production of TBb hypoallergens and to allergen-specific immunotherapy for tartary buckwheat allergy.

  9. Improving wheat to remove coeliac epitopes but retain functionality.

    Science.gov (United States)

    Shewry, Peter R; Tatham, Arthur S

    2016-01-01

    Coeliac disease is an intolerance triggered by the ingestion of wheat gluten proteins. It is of increasing concern to consumers and health professionals as its incidence appears to be increasing. The amino acid sequences in gluten proteins that are responsible for triggering responses in sensitive individuals have been identified showing that they vary in distribution among and between different groups of gluten proteins. Conventional breeding may therefore be used to select for gluten protein fractions with lower contents of coeliac epitopes. Molecular breeding approaches can also be used to specifically down-regulate coeliac-toxic proteins or mutate coeliac epitopes within individual proteins. A combination of these approaches may therefore be used to develop a "coeliac-safe" wheat. However, this remains a formidable challenge due to the complex multigenic control of gluten protein composition. Furthermore, any modified wheats must retain acceptable properties for making bread and other processed foods. Not surprisingly, such coeliac-safe wheats have not yet been developed despite over a decade of research.

  10. Antibody Recognition of a Highly Conserved Influenza Virus Epitope

    Energy Technology Data Exchange (ETDEWEB)

    Ekiert, Damian C.; Bhabha, Gira; Elsliger, Marc-André; Friesen, Robert H.E.; Jongeneelen, Mandy; Throsby, Mark; Goudsmit, Jaap; Wilson, Ian A.; Scripps; Crucell

    2009-05-21

    Influenza virus presents an important and persistent threat to public health worldwide, and current vaccines provide immunity to viral isolates similar to the vaccine strain. High-affinity antibodies against a conserved epitope could provide immunity to the diverse influenza subtypes and protection against future pandemic viruses. Cocrystal structures were determined at 2.2 and 2.7 angstrom resolutions for broadly neutralizing human antibody CR6261 Fab in complexes with the major surface antigen (hemagglutinin, HA) from viruses responsible for the 1918 H1N1 influenza pandemic and a recent lethal case of H5N1 avian influenza. In contrast to other structurally characterized influenza antibodies, CR6261 recognizes a highly conserved helical region in the membrane-proximal stem of HA1 and HA2. The antibody neutralizes the virus by blocking conformational rearrangements associated with membrane fusion. The CR6261 epitope identified here should accelerate the design and implementation of improved vaccines that can elicit CR6261-like antibodies, as well as antibody-based therapies for the treatment of influenza.

  11. Selection of a peptide mimicking neutralization epitope of hepatitis E virus with phage peptide display technology

    Institute of Scientific and Technical Information of China (English)

    Ying Gu; Jun Zhang; Ying-Bing Wang; Shao-Wei Li; Hai-Jie Yang; Wen-Xin Luo; Ning-Shao Xia

    2004-01-01

    AIM: To select the peptide mimicking the neutralization epitope of hepatitis E virus which bound to non-type-specific and conformational monoclonal antibodies (mAbs) 8C11 and 8H3 fromed 7-peptide phage display library, and expressed the peptide recombinant with HBcAg in E.coli, and to observe whether the recombinant HBcAg could still form virus like particle (VLP) and to test the activation of the recombinant polyprotein and chemo-synthesized peptide that was selected by mAb 8H3.METHODS: 8C11 and 8H3 were used to screen for binding peptides through a 7-peptide phage display library. After 4rounds of panning, monoclonal phages were selected and sequenced. The obtained dominant peptide coding sequences was then synthesized and inserted into amino acid 78 to 83 of hepatitis B core antigen (HBcAg), and then expressed in E. coli. Activity of the recombinant proteins was detected by Western blotting, VLPs of the recombinant polyproteins were tested by transmission electron microscopy and binding activity of the chemo-synthesized peptide was confirmed by BIAcore biosensor.RESULTS: Twenty-one positive monoclonal phages (10for 8CL1, and 11 for 8H3) were selected and the inserted fragments were sequenced. The DNA sequence coding for the obtained dominant peptides 8C11 (N′-His-Pro-Thr-LeuLeu-Arg-Ile-C′, named 8C11A) and 8H3 (N′-Ser-Ile-LeuPro- Tyr-Pro-Tyr-C′, named 8H3A) were then synthesized and cloned to the HBcAg vector, then expressed in E. coli.The recombinant proteins aggregated into homodimer or polymer on SDS-PAGE, and could bind to mAb 8C11 and 8H3 in Western blotting. At the same time, the recombinant polyprotein could form virus like particles (VLPs), which could be visualized on electron micrograph. The dominant peptide 8H3A selected by mAb 8H3 was further chemosynthesized, and its binding to mAb 8H3 could be detected by BIAcore biosensor.CONCLUSION: These results implicate that conformational neutralizing epitope can be partially modeled by a short

  12. ArrayPitope: Automated Analysis of Amino Acid Substitutions for Peptide Microarray-Based Antibody Epitope Mapping

    DEFF Research Database (Denmark)

    Hansen, Christian Skjødt; Østerbye, Thomas; Marcatili, Paolo

    2017-01-01

    Identification of epitopes targeted by antibodies (B cell epitopes) is of critical importance for the development of many diagnostic and therapeutic tools. For clinical usage, such epitopes must be extensively characterized in order to validate specificity and to document potential cross-reactivi...

  13. ElliPro: a new structure-based tool for the prediction of antibody epitopes

    Directory of Open Access Journals (Sweden)

    Fusseder Nicholas

    2008-12-01

    Full Text Available Abstract Background Reliable prediction of antibody, or B-cell, epitopes remains challenging yet highly desirable for the design of vaccines and immunodiagnostics. A correlation between antigenicity, solvent accessibility, and flexibility in proteins was demonstrated. Subsequently, Thornton and colleagues proposed a method for identifying continuous epitopes in the protein regions protruding from the protein's globular surface. The aim of this work was to implement that method as a web-tool and evaluate its performance on discontinuous epitopes known from the structures of antibody-protein complexes. Results Here we present ElliPro, a web-tool that implements Thornton's method and, together with a residue clustering algorithm, the MODELLER program and the Jmol viewer, allows the prediction and visualization of antibody epitopes in a given protein sequence or structure. ElliPro has been tested on a benchmark dataset of discontinuous epitopes inferred from 3D structures of antibody-protein complexes. In comparison with six other structure-based methods that can be used for epitope prediction, ElliPro performed the best and gave an AUC value of 0.732, when the most significant prediction was considered for each protein. Since the rank of the best prediction was at most in the top three for more than 70% of proteins and never exceeded five, ElliPro is considered a useful research tool for identifying antibody epitopes in protein antigens. ElliPro is available at http://tools.immuneepitope.org/tools/ElliPro. Conclusion The results from ElliPro suggest that further research on antibody epitopes considering more features that discriminate epitopes from non-epitopes may further improve predictions. As ElliPro is based on the geometrical properties of protein structure and does not require training, it might be more generally applied for predicting different types of protein-protein interactions.

  14. Epitope-driven DNA vaccine design employing immunoinformatics against B-cell lymphoma: a biotech's challenge.

    Science.gov (United States)

    Iurescia, Sandra; Fioretti, Daniela; Fazio, Vito Michele; Rinaldi, Monica

    2012-01-01

    DNA vaccination has been widely explored to develop new, alternative and efficient vaccines for cancer immunotherapy. DNA vaccines offer several benefits such as specific targeting, use of multiple genes to enhance immunity and reduced risk compared to conventional vaccines. Rapid developments in molecular biology and immunoinformatics enable rational design approaches. These technologies allow construction of DNA vaccines encoding selected tumor antigens together with molecules to direct and amplify the desired effector pathways, as well as highly targeted vaccines aimed at specific epitopes. Reliable predictions of immunogenic T cell epitope peptides are crucial for rational vaccine design and represent a key problem in immunoinformatics. Computational approaches have been developed to facilitate the process of epitope detection and show potential applications to the immunotherapeutic treatment of cancer. In this review a number of different epitope prediction methods are briefly illustrated and effective use of these resources to support experimental studies is described. Epitope-driven vaccine design employs these bioinformatics algorithms to identify potential targets of vaccines against cancer. In this paper the selection of T cell epitopes to develop epitope-based vaccines, the need for CD4(+) T cell help for improved vaccines and the assessment of vaccine performance against tumor are reviewed. We focused on two applications, namely prediction of novel T cell epitopes and epitope enhancement by sequence modification, and combined rationale design with bioinformatics for creation of new synthetic mini-genes. This review describes the development of epitope-based DNA vaccines and their antitumor effects in preclinical research against B-cell lymphoma, corroborating the usefulness of this platform as a potential tool for cancer therapy. Achievements in the field of DNA vaccines allow to overcome hurdles to clinical translation. In a scenario where the vaccine

  15. Identification of a dual-specific T cell epitope of the hemagglutinin antigen of an h5 avian influenza virus in chickens.

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    Hamid R Haghighi

    Full Text Available Avian influenza viruses (AIV of the H5N1 subtype have caused morbidity and mortality in humans. Although some migratory birds constitute the natural reservoir for this virus, chickens may play a role in transmission of the virus to humans. Despite the importance of avian species in transmission of AIV H5N1 to humans, very little is known about host immune system interactions with this virus in these species. The objective of the present study was to identify putative T cell epitopes of the hemagglutinin (HA antigen of an H5 AIV in chickens. Using an overlapping peptide library covering the HA protein, we identified a 15-mer peptide, H5(246-260, within the HA1 domain which induced activation of T cells in chickens immunized against the HA antigen of an H5 virus. Furthermore, H5(246-260 epitope was found to be presented by both major histocompatibility complex (MHC class I and II molecules, leading to activation of CD4+ and CD8+ T cell subsets, marked by proliferation and expression of interferon (IFN-gamma by both of these cell subsets as well as the expression of granzyme A by CD8+ T cells. This is the first report of a T cell epitope of AIV recognized by chicken T cells. Furthermore, this study extends the previous finding of the existence of dual-specific epitopes in other species to chickens. Taken together, these results elucidate some of the mechanisms of immune response to AIV in chickens and provide a platform for creation of rational vaccines against AIV in this species.

  16. Down-regulation of αGal epitopes by co-transfection of α1,3-galactosidase gene and α1,2-fucosyltransferase gene

    Institute of Scientific and Technical Information of China (English)

    GONG Feng; ZHANG Yangpei; JIA Yanjun; WANG Yingli; TAN Yingxia; TIAN Shuguang

    2005-01-01

    The polycarbohydrate structure of Galα1- 3Galβ1-4GluNAc-R (known as αGal epitopes of xenoantigen), produced by α1-3-galactosyltransferase (α1,3-GT) in the course of animal development, is the major xenoantigen on the cell surface of porcine which causes hyperacute rejection in pig-to-human xenotransplantation. Alpha-1,3-galactosi- dase (AGL), a hydrolytic enzyme, can remove the terminal α-1,3-galactosyl from the Galα1-3Galβ1-4GluNAc-R structure resulting in cleaning αGal epitopes from the porcine cells. Alpha-1,2-fucosyltransferase (HT) can modify the surface carbohydrate phenotype of porcine cells, bringing about reduction of αGal epitopes expression. In this study, human AGL and HT gene were co-transfected to porcine fetal fibroblast (PFFb) in equimolar concentration to reduce the xenoantigen. Gene and protein of hAGL and HT were both detected to express at high level by RT-PCR and Western blot, respectively. There was an 84% reduction in αGal xenoantigen and an 82% increase in H antigen as assayed by flow cytometry in the AGL and HT gene co-transfected PFFb. The number and morphology of transgenic PFFb chromosome were normal. Findings indicate that Galα1-3Gal epitopes of PFFb could be down regulated by AGL and HT co-transfection without deleterious effects on the chromosomal profile of the transgenic cell.

  17. Antigenic topology of the P29 surface lipoprotein of Mycoplasma fermentans: differential display of epitopes results in high-frequency phase variation.

    Science.gov (United States)

    Theiss, P; Karpas, A; Wise, K S

    1996-05-01

    Antibodies to P29, a major lipid-modified surface protein of Mycoplasma fermentans, reveal phase variation of surface epitopes occurring with high frequency in clonal lineages of the organism. This occurs despite continuous expression of the entire epitope-bearing P29 product (detected by Western immunoblotting) and contrasts with phase variation of other surface antigens mediated by differential expression of proteins. To understand the structure and antigenic topology of P29, the single-copy p29 gene from strain PG18 was cloned and sequenced. The gene encodes a prolipoprotein containing a signal sequence predicted to be modified with lipid and cleaved at the N-terminal Cys-1 residue of the mature P29 lipoprotein. The remaining 218-residue hydrophilic sequence of P29 is predicted to be located external to the single plasma membrane. Additional Cys residues at positions 91 and 128 in the mature protein were shown to form a 36-residue disulfide loop by selectively labeling sulfhydryl groups that were liberated only after chemical reduction of monomeric P29. Two nearly identical charged amino acid sequences occurred in P29, within the disulfide loop and upstream of this structure. Two distinct epitopes binding different monoclonal antibodies were associated with opposite ends of the P29 protein, by mapping products expressed in Escherichia coli from PCR-generated 3' deletion mutations of the p29 gene. Each monoclonal antibody detected high-frequency and noncoordinate changes in accessibility of the corresponding epitopes in colony immunoblots of clonal variants, yet sequencing of the p29 gene from these variants and analysis of disulfide bonds revealed no associated changes in the primary sequence or disulfide loop structure of P29. These results suggest that P29 surface epitope variation may involve masking of selected regions of P29, possibly by other surface components undergoing phase variation by differential expression. Differential masking may be an important

  18. A novel HLA-B18 restricted CD8+ T cell epitope is efficiently cross-presented by dendritic cells from soluble tumor antigen.

    Directory of Open Access Journals (Sweden)

    Rona Y Zhao

    Full Text Available NY-ESO-1 has been a major target of many immunotherapy trials because it is expressed by various cancers and is highly immunogenic. In this study, we have identified a novel HLA-B*1801-restricted CD8(+ T cell epitope, NY-ESO-1(88-96 (LEFYLAMPF and compared its direct- and cross-presentation to that of the reported NY-ESO-1(157-165 epitope restricted to HLA-A*0201. Although both epitopes were readily cross-presented by DCs exposed to various forms of full-length NY-ESO-1 antigen, remarkably NY-ESO-1(88-96 is much more efficiently cross-presented from the soluble form, than NY-ESO-1(157-165. On the other hand, NY-ESO-1(157-165 is efficiently presented by NY-ESO-1-expressing tumor cells and its presentation was not enhanced by IFN-γ treatment, which induced immunoproteasome as demonstrated by Western blots and functionally a decreased presentation of Melan A(26-35; whereas NY-ESO-1(88-96 was very inefficiently presented by the same tumor cell lines, except for one that expressed high level of immunoproteasome. It was only presented when the tumor cells were first IFN-γ treated, followed by infection with recombinant vaccinia virus encoding NY-ESO-1, which dramatically increased NY-ESO-1 expression. These data indicate that the presentation of NY-ESO-1(88-96 is immunoproteasome dependent. Furthermore, a survey was conducted on multiple samples collected from HLA-B18(+ melanoma patients. Surprisingly, all the detectable responses to NY-ESO-1(88-96 from patients, including those who received NY-ESO-1 ISCOMATRIX™ vaccine were induced spontaneously. Taken together, these results imply that some epitopes can be inefficiently presented by tumor cells although the corresponding CD8(+ T cell responses are efficiently primed in vivo by DCs cross-presenting these epitopes. The potential implications for cancer vaccine strategies are further discussed.

  19. Detailed Analysis of the Expression of an Alpha-gliadin Promoter and the Deposition of Alpha-gliadin Protein During Wheat Grain Development

    NARCIS (Netherlands)

    Herpen, van T.W.J.M.; Riley, M.; Sparks, C.; Jones, H.D.; Gritsch, C.; Dekking, E.H.; Hamer, R.J.; Bosch, H.J.; Salentijn, E.M.J.; Smulders, M.J.M.; Shewry, P.R.; Gilissen, L.J.W.J.

    2008-01-01

    Background and Aims: Alpha-gliadin proteins are important for the industrial quality of bread wheat flour, but they also contain many epitopes that can trigger celiac (c¿liac) disease (CD). The B-genome-encoded -gliadin genes, however, contain very few epitopes. Controlling -gliadin gene expression

  20. Carbohydrate Microarrays Identify Blood Group Precursor Cryptic Epitopes as Potential Immunological Targets of Breast Cancer

    Directory of Open Access Journals (Sweden)

    Denong Wang

    2015-01-01

    Full Text Available Using carbohydrate microarrays, we explored potential natural ligands of antitumor monoclonal antibody HAE3. This antibody was raised against a murine mammary tumor antigen but was found to cross-react with a number of human epithelial tumors in tissues. Our carbohydrate microarray analysis reveals that HAE3 is specific for an O-glycan cryptic epitope that is normally hidden in the cores of blood group substances. Using HAE3 to screen tumor cell surface markers by flow cytometry, we found that the HAE3 glycoepitope, gpHAE3, was highly expressed by a number of human breast cancer cell lines, including some triple-negative cancers that lack the estrogen, progesterone, and Her2/neu receptors. Taken together, we demonstrate that HAE3 recognizes a conserved cryptic glycoepitope of blood group precursors, which is nevertheless selectively expressed and surface-exposed in certain breast tumor cells. The potential of this class of O-glycan cryptic antigens in breast cancer subtyping and targeted immunotherapy warrants further investigation.

  1. Identification of B cell epitopes of alcohol dehydrogenase allergen of Curvularia lunata.

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    Smitha Nair

    Full Text Available BACKGROUND/OBJECTIVE: Epitope identification assists in developing molecules for clinical applications and is useful in defining molecular features of allergens for understanding structure/function relationship. The present study was aimed to identify the B cell epitopes of alcohol dehydrogenase (ADH allergen from Curvularia lunata using in-silico methods and immunoassay. METHOD: B cell epitopes of ADH were predicted by sequence and structure based methods and protein-protein interaction tools while T cell epitopes by inhibitory concentration and binding score methods. The epitopes were superimposed on a three dimensional model of ADH generated by homology modeling and analyzed for antigenic characteristics. Peptides corresponding to predicted epitopes were synthesized and immunoreactivity assessed by ELISA using individual and pooled patients' sera. RESULT: The homology model showed GroES like catalytic domain joined to Rossmann superfamily domain by an alpha helix. Stereochemical quality was confirmed by Procheck which showed 90% residues in most favorable region of Ramachandran plot while Errat gave a quality score of 92.733%. Six B cell (P1-P6 and four T cell (P7-P10 epitopes were predicted by a combination of methods. Peptide P2 (epitope P2 showed E(X(2GGP(X(3KKI conserved pattern among allergens of pathogenesis related family. It was predicted as high affinity binder based on electronegativity and low hydrophobicity. The computational methods employed were validated using Bet v 1 and Der p 2 allergens where 67% and 60% of the epitope residues were predicted correctly. Among B cell epitopes, Peptide P2 showed maximum IgE binding with individual and pooled patients' sera (mean OD 0.604±0.059 and 0.506±0.0035, respectively followed by P1, P4 and P3 epitopes. All T cell epitopes showed lower IgE binding. CONCLUSION: Four B cell epitopes of C. lunata ADH were identified. Peptide P2 can serve as a potential candidate for diagnosis of allergic

  2. Protection against P. aeruginosa with an adenovirus vector containing an OprF epitope in the capsid.

    Science.gov (United States)

    Worgall, Stefan; Krause, Anja; Rivara, Michael; Hee, Kyung-Kim; Vintayen, Enrico V; Hackett, Neil R; Roelvink, Peter W; Bruder, Joseph T; Wickham, Thomas J; Kovesdi, Imre; Crystal, Ronald G

    2005-05-01

    Pseudomonas aeruginosa is an important opportunistic pathogen that can cause chronic and often life-threatening infections of the respiratory tract, particularly in individuals with cystic fibrosis (CF). Because infections with P. aeruginosa remain the major cause of the high morbidity and mortality of CF, a vaccine against P. aeruginosa would be very useful for preventing this disorder. The outer membrane protein F (OprF) of P. aeruginosa is a promising vaccine candidate and various B cell epitopes within OprF have been identified. Given that adenovirus (Ad) vectors have strong immunogenic potential and can function as adjuvants for genetic vaccines, the present study evaluates the immunogenic and protective properties of a novel replication-deficient Ad vector in which the Ad hexon protein was modified to include a 14-amino acid epitope of P. aeruginosa OprF (Epi8) in loop 1 of the hypervariable region 5 of the hexon (AdZ.Epi8). Immunization of C57BL/6 mice with AdZ.Epi8 resulted in detectable serum anti-P. aeruginosa and anti-OprF humoral responses. These responses were haplotype dependent, with higher serum anti-OprF titers in CBA mice than in BALB/c or C57BL/6 mice. AdZ.Epi8 induced Epi8-specific IFN-gamma-positive CD4 and CD8 T cell responses and resulted in protection against a lethal pulmonary challenge with agar-encapsulated P. aeruginosa. Importantly, repeated administration of AdZ.Epi8 resulted in boosting of the anti-OprF humoral and anti-Epi8 cellular response, whereas no boosting effect was present in the response against the transgene beta-galactosidase. These observations suggest that Ad vectors expressing pathogen epitopes in their capsid will protect against an extracellular pathogen and will allow boosting of the epitope-specific humoral response with repeated administration, a strategy that should prove useful in developing Ad vectors as vaccines where humoral immunity will be protective.

  3. Engineering Recombinant Reoviruses To Display gp41 Membrane-Proximal External-Region Epitopes from HIV-1

    Science.gov (United States)

    Boehme, Karl W.; Ikizler, Mine'; Iskarpatyoti, Jason A.; Wetzel, J. Denise; Willis, Jordan; Crowe, James E.; LaBranche, Celia C.; Montefiori, David C.

    2016-01-01

    . Antibodies that neutralize genetically diverse strains of HIV-1 bind to discrete regions of the envelope glycoproteins, including the gp41 MPER. We engineered recombinant reoviruses that displayed MPER epitopes in attachment protein σ1 (REO-MPER vectors). The REO-MPER vectors replicated with wild-type efficiency, were genetically stable, and retained native antigenicity. However, we did not detect HIV-1-specific immune responses following inoculation of the REO-MPER vectors into small animals. This work provides proof of principle for engineering reovirus to express antigenic epitopes and illustrates the difficulty in eliciting MPER-specific immune responses. PMID:27303748

  4. PRRSV非结构蛋白2-半胱氨酸结构域的原核表达及抗原表位预测%The Gene Clone, Prokaryotic Expression and Antigen Epitopes Prediction of the Cysteine Domain of Nonstructural Protein 2 of PRRSV

    Institute of Scientific and Technical Information of China (English)

    周胜; 高歌; 孙荡; 鲍梦雅; 茅翔

    2012-01-01

    为了获得PRRSV非结构蛋白2-半胱氨酸结构域的高纯度原核表达蛋白以及了解该蛋白的抗原表位,试验以从PRRSV-VR2332毒株上提取的病毒全基因组RNA为模板,通过RT-PCR扩增非结构蛋白2-半胱氨酸结构域(Nsp2pro)基因,连接构建原核表达载体SUMO-Nsp2pro,转入宿主茵Rosetta2,经IPTG诱导,获得高浓度的可溶蛋白Nsp2pro,经亲和层析His-Bind后得到高纯度的目的蛋白。同时,运用生物信息学方法对Nsp2pro二级结构及抗原表位进行预测。结果显示,Nsp2pro的α螺旋及β折叠区域较多,转角区域较少,结构较为复杂,位于蛋白分子表面且亲水的区段很可能是B细胞表位的优势区段。获得较高纯度的蛋白,将为后续进一步研究Nsp2pro基因编码的蛋白在病毒复制过程中的作用奠定基础。%In order to acquire highly purified protein of cysteine domain of nonstructural protein 2 of PRRSV and acquaintance its antigen epiopes, the experiment were conducted with the genome extracted from PRRSV-VR2332 and got the gene of the cysteine domain of nonstructural protein 2 of PRRSV(Nsp2pro)by RT-PCR. Then,a recombinant plasmid named SUMO-Nsp2pro had been constructed, and then transformed SUMO-Nsp2pro into Rosetta2. Therefore,a large amount of Nsp2pro was obtained by IPTG. In the end,Nsp2pro was purified by His-Bind affinity chromatography,meanwhile predicted the second structure and antigen epiopes through means of hio-information,the results showed that Nsp2pro owned a great many Alpha regions and Beta regions while few of turn regions. The antigen epitopes were located in hydrolicity regions possibly. The obtained high purified protein would provide foundations for the further researches on Nsp2pro gene.

  5. Epitope-based recombinant diagnostic antigen to distinguish natural infection from vaccination with hepatitis A virus vaccines.

    Science.gov (United States)

    Su, Qiudong; Guo, Minzhuo; Jia, Zhiyuan; Qiu, Feng; Lu, Xuexin; Gao, Yan; Meng, Qingling; Tian, Ruiguang; Bi, Shengli; Yi, Yao

    2016-07-01

    Hepatitis A virus (HAV) infection can stimulate the production of antibodies to structural and non-structural proteins of the virus. However, vaccination with an inactivated or attenuated HAV vaccine produces antibodies mainly against structural proteins, whereas no or very limited antibodies are produced against the non-structural proteins. Current diagnostic assays to determine exposure to HAV, such as the Abbott HAV AB test, detect antibodies only to the structural proteins and so are not able to distinguish a natural infection from vaccination with an inactivated or attenuated virus. Here, we constructed a recombinant tandem multi-epitope diagnostic antigen (designated 'H1') based on the immune-dominant epitopes of the non-structural proteins of HAV to distinguish the two situations. H1 protein expressed in Escherichia coli and purified by affinity and anion exchange chromatography was applied in a double-antigen sandwich ELISA for the detection of anti-non-structural HAV proteins, which was confirmed to distinguish a natural infection from vaccination with an inactivated or attenuated HAV vaccine.

  6. A Nanoparticle Based Sp17 Peptide Vaccine Exposes New Immuno-Dominant and Species Cross-reactive B Cell Epitopes

    Directory of Open Access Journals (Sweden)

    Sue D. Xiang

    2015-10-01

    Full Text Available Sperm protein antigen 17 (Sp17, expressed in primary as well as in metastatic lesions in >83% of patients with ovarian cancer, is a promising ovarian cancer vaccine candidate. Herein we describe the formulation of nanoparticle based vaccines based on human Sp17 (hSp17 sequence derived peptides, and map the immuno-dominant T cell and antibody epitopes induced using such formulations. The primary T and B cell immuno-dominant region within Sp17 was found to be the same when using biocompatible nanoparticle carriers or the conventional “mix-in” pro-inflammatory adjuvant CpG, both mapping to amino acids (aa 111–142. However, delivery of hSp17111–142 as a nanoparticle conjugate promoted a number of new properties, changing the dominant antibody isotype induced from IgG2a to IgG1 and the fine specificity of the B cell epitopes within hSp17111–142, from an immuno-dominant region 134–142 aa for CpG, to region 121–138 aa for nanoparticles. Associated with this change in specificity was a substantial increase in antibody cross-reactivity between mouse and human Sp17. These results indicate conjugation of antigen to nanoparticles can have major effects on fine antigen specificity, which surprisingly could be beneficially used to increase the cross-reactivity of antibody responses.

  7. Combining computer algorithms with experimental approaches permits the rapid and accurate identification of T cell epitopes from defined antigens.

    Science.gov (United States)

    Schirle, M; Weinschenk, T; Stevanović, S

    2001-11-01

    The identification of T cell epitopes from immunologically relevant antigens remains a critical step in the development of vaccines and methods for monitoring of T cell responses. This review presents an overview of strategies that employ computer algorithms for the selection of candidate peptides from defined proteins and subsequent verification of their in vivo relevance by experimental approaches. Several computer algorithms are currently being used for epitope prediction of various major histocompatibility complex (MHC) class I and II molecules, based either on the analysis of natural MHC ligands or on the binding properties of synthetic peptides. Moreover, the analysis of proteasomal digests of peptides and whole proteins has led to the development of algorithms for the prediction of proteasomal cleavages. In order to verify the generation of the predicted peptides during antigen processing in vivo as well as their immunogenic potential, several experimental approaches have been pursued in the recent past. Mass spectrometry-based bioanalytical approaches have been used specifically to detect predicted peptides among isolated natural ligands. Other strategies employ various methods for the stimulation of primary T cell responses against the predicted peptides and subsequent testing of the recognition pattern towards target cells that express the antigen.

  8. Elicitation of neutralizing antibodies directed against CD4-induced epitope(s using a CD4 mimetic cross-linked to a HIV-1 envelope glycoprotein.

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    Antu K Dey

    Full Text Available The identification of HIV-1 envelope glycoprotein (Env structures that can generate broadly neutralizing antibodies (BNAbs is pivotal to the development of a successful vaccine against HIV-1 aimed at eliciting effective humoral immune responses. To that end, the production of novel Env structure(s that might induce BNAbs by presentation of conserved epitopes, which are otherwise occluded, is critical. Here, we focus on a structure that stabilizes Env in a conformation representative of its primary (CD4 receptor-bound state, thereby exposing highly conserved "CD4 induced" (CD4i epitope(s known to be important for co-receptor binding and subsequent virus infection. A CD4-mimetic miniprotein, miniCD4 (M64U1-SH, was produced and covalently complexed to recombinant, trimeric gp140 envelope glycoprotein (gp140 using site-specific disulfide linkages. The resulting gp140-miniCD4 (gp140-S-S-M64U1 complex was recognized by CD4i antibodies and the HIV-1 co-receptor, CCR5. The gp140-miniCD4 complex elicited the highest titers of CD4i binding antibodies as well as enhanced neutralizing antibodies against Tier 1 viruses as compared to gp140 protein alone following immunization of rabbits. Neutralization against HIV-2(7312/V434M and additional serum mapping confirm the specific elicitation of antibodies directed to the CD4i epitope(s. These results demonstrate the utility of structure-based approach in improving immunogenic response against specific region, such as the CD4i epitope(s here, and its potential role in vaccine application.

  9. Rational design of antibodies targeting specific epitopes within intrinsically disordered proteins

    Science.gov (United States)

    Sormanni, Pietro; Aprile, Francesco A.; Vendruscolo, Michele

    2015-01-01

    Antibodies are powerful tools in life sciences research, as well as in diagnostic and therapeutic applications, because of their ability to bind given molecules with high affinity and specificity. Using current methods, however, it is laborious and sometimes difficult to generate antibodies to target specific epitopes within a protein, in particular if these epitopes are not effective antigens. Here we present a method to rationally design antibodies to enable them to bind virtually any chosen disordered epitope in a protein. The procedure consists in the sequence-based design of one or more complementary peptides targeting a selected disordered epitope and the subsequent grafting of such peptides on an antibody scaffold. We illustrate the method by designing six single-domain antibodies to bind different epitopes within three disease-related intrinsically disordered proteins and peptides (α-synuclein, Aβ42, and IAPP). Our results show that all these designed antibodies bind their targets with good affinity and specificity. As an example of an application, we show that one of these antibodies inhibits the aggregation of α-synuclein at substoichiometric concentrations and that binding occurs at the selected epitope. Taken together, these results indicate that the design strategy that we propose makes it possible to obtain antibodies targeting given epitopes in disordered proteins or protein regions. PMID:26216991

  10. Rational design of antibodies targeting specific epitopes within intrinsically disordered proteins.

    Science.gov (United States)

    Sormanni, Pietro; Aprile, Francesco A; Vendruscolo, Michele

    2015-08-11

    Antibodies are powerful tools in life sciences research, as well as in diagnostic and therapeutic applications, because of their ability to bind given molecules with high affinity and specificity. Using current methods, however, it is laborious and sometimes difficult to generate antibodies to target specific epitopes within a protein, in particular if these epitopes are not effective antigens. Here we present a method to rationally design antibodies to enable them to bind virtually any chosen disordered epitope in a protein. The procedure consists in the sequence-based design of one or more complementary peptides targeting a selected disordered epitope and the subsequent grafting of such peptides on an antibody scaffold. We illustrate the method by designing six single-domain antibodies to bind different epitopes within three disease-related intrinsically disordered proteins and peptides (α-synuclein, Aβ42, and IAPP). Our results show that all these designed antibodies bind their targets with good affinity and specificity. As an example of an application, we show that one of these antibodies inhibits the aggregation of α-synuclein at substoichiometric concentrations and that binding occurs at the selected epitope. Taken together, these results indicate that the design strategy that we propose makes it possible to obtain antibodies targeting given epitopes in disordered proteins or protein regions.

  11. Epitope specific T-cell responses against influenza A in a healthy population.

    Science.gov (United States)

    Savic, Miloje; Dembinski, Jennifer L; Kim, Yohan; Tunheim, Gro; Cox, Rebecca J; Oftung, Fredrik; Peters, Bjoern; Mjaaland, Siri

    2016-02-01

    Pre-existing human CD4(+) and CD8(+) T-cell-mediated immunity may be a useful correlate of protection against severe influenza disease. Identification and evaluation of common epitopes recognized by T cells with broad cross-reactivity is therefore important to guide universal influenza vaccine development, and to monitor immunological preparedness against pandemics. We have retrieved an optimal combination of MHC class I and class II restricted epitopes from the Immune Epitope Database (www.iedb.org), by defining a fitness score function depending on prevalence, sequence conservancy and HLA super-type coverage. Optimized libraries of CD4(+) and CD8(+) T-cell epitopes were selected from influenza antigens commonly present in seasonal and pandemic influenza strains from 1934 to 2009. These epitope pools were used to characterize human T-cell responses in healthy donors using interferon-γ ELISPOT assays. Upon stimulation, significant CD4(+) and CD8(+) T-cell responses were induced, primarily recognizing epitopes from the conserved viral core proteins. Furthermore, the CD4(+) and CD8(+) T cells were phenotypically characterized regarding functionality, cytotoxic potential and memory phenotype using flow cytometry. Optimized sets of T-cell peptide epitopes may be a useful tool to monitor the efficacy of clinical trials, the immune status of a population to predict immunological preparedness against pandemics, as well as being candidates for universal influenza vaccines.

  12. PepMapper: a collaborative web tool for mapping epitopes from affinity-selected peptides.

    Directory of Open Access Journals (Sweden)

    Wenhan Chen

    Full Text Available Epitope mapping from affinity-selected peptides has become popular in epitope prediction, and correspondingly many Web-based tools have been developed in recent years. However, the performance of these tools varies in different circumstances. To address this problem, we employed an ensemble approach to incorporate two popular Web tools, MimoPro and Pep-3D-Search, together for taking advantages offered by both methods so as to give users more options for their specific purposes of epitope-peptide mapping. The combined operation of Union finds as many associated peptides as possible from both methods, which increases sensitivity in finding potential epitopic regions on a given antigen surface. The combined operation of Intersection achieves to some extent the mutual verification by the two methods and hence increases the likelihood of locating the genuine epitopic region on a given antigen in relation to the interacting peptides. The Consistency between Intersection and Union is an indirect sufficient condition to assess the likelihood of successful peptide-epitope mapping. On average from 27 tests, the combined operations of PepMapper outperformed either MimoPro or Pep-3D-Search alone. Therefore, PepMapper is another multipurpose mapping tool for epitope prediction from affinity-selected peptides. The Web server can be freely accessed at: http://informatics.nenu.edu.cn/PepMapper/

  13. Multipin peptide libraries for antibody and receptor epitope screening and characterization.

    Science.gov (United States)

    Tribbick, Gordon

    2002-09-01

    It has been nearly 15 years since the papers describing the fully systematic epitope mapping approach both for the so-called "continuous" epitopes [Proc. Natl. Acad. Sci. U. S. A. 81 (1984) 3998] and "discontinuous" epitopes [Mol. Immunol. 23 (1986) 709] were published. These seminal papers laid the conceptual foundation for all subsequent developments where a combinatorial approach is applied. Dr. Mario Geysen, the 2000 Kilby Laureate, can certainly lay claim to be the "father of combinatorial chemistry" (http://www.kilby.org/laureates.htm). In this review, I will focus on the aspects of the Multipin technology as they apply to antibody and receptor epitope mapping. Much of what will be presented applies equally well to other applications where peptide libraries (PepSets) and combinatorial approaches are used [Rodda, S.J., 1996. T-cell epitope mapping with synthetic peptides and peripheral blood mononuclear cells. In: Morris, G.E. (Eds.), Methods in Molecular Biology, Vol. 66: Epitope Mapping Protocols. Humana Press, Totowa, NJ, Chap. 30, p. 363; Int. J. Pept. Protein Res. 42 (1993) 384; J. Biol. Chem. 271 (1996) 5603]. Factors and techniques that influence the use of the Multipin method for successful epitope mapping will be presented.

  14. A Comparison of Epitope Repertoires Associated with Myasthenia Gravis in Humans and Nonhuman Hosts

    Directory of Open Access Journals (Sweden)

    Kerrie Vaughan

    2012-01-01

    Full Text Available Here we analyzed the molecular targets associated with myasthenia gravis (MG immune responses, enabled by an immune epitope database (IEDB inventory of approximately 600 MG-related epitopes derived from 175 references. The vast majority of epitopes were derived from the α-subunit of human AChR suggesting that other MG-associated autoantigens should be investigated further. Human α-AChR was mostly characterized in humans, whereas reactivity primarily to T. californica AChR was examined in animal models. While the fine specificity of T-cell response was similar in the two systems, substantial antibody reactivity to the C-terminus was detected in the nonhuman system, but not in humans. Further analysis showed that the reactivity of nonhuman hosts to the C-terminus was eliminated when data were restricted to hosts tested in the context of autoimmune disease (spontaneous or induced, demonstrating that the epitopes recognized in humans and animals were shared when disease was present. Finally, we provided data subsets relevant to particular applications, including those associated with HLA typing or restriction, sets of epitopes recognized by monoclonal antibodies, and epitopes associated with modulation of immunity or disease. In conclusion, this analysis highlights gaps, differences, and similarities in the epitope repertoires of humans and animal models.

  15. In Vivo Validation of Predicted and Conserved T Cell Epitopes in a Swine Influenza Model

    Science.gov (United States)

    Gutiérrez, Andres H.; Loving, Crystal; Moise, Leonard; Terry, Frances E.; Brockmeier, Susan L.; Hughes, Holly R.; Martin, William D.; De Groot, Anne S.

    2016-01-01

    Swine influenza is a highly contagious respiratory viral infection in pigs that is responsible for significant financial losses to pig farmers annually. Current measures to protect herds from infection include: inactivated whole-virus vaccines, subunit vaccines, and alpha replicon-based vaccines. As is true for influenza vaccines for humans, these strategies do not provide broad protection against the diverse strains of influenza A virus (IAV) currently circulating in U.S. swine. Improved approaches to developing swine influenza vaccines are needed. Here, we used immunoinformatics tools to identify class I and II T cell epitopes highly conserved in seven representative strains of IAV in U.S. swine and predicted to bind to Swine Leukocyte Antigen (SLA) alleles prevalent in commercial swine. Epitope-specific interferon-gamma (IFNγ) recall responses to pooled peptides and whole virus were detected in pigs immunized with multi-epitope plasmid DNA vaccines encoding strings of class I and II putative epitopes. In a retrospective analysis of the IFNγ responses to individual peptides compared to predictions specific to the SLA alleles of cohort pigs, we evaluated the predictive performance of PigMatrix and demonstrated its ability to distinguish non-immunogenic from immunogenic peptides and to identify promiscuous class II epitopes. Overall, this study confirms the capacity of PigMatrix to predict immunogenic T cell epitopes and demonstrate its potential for use in the design of epitope-driven vaccines for swine. Additional studies that match the SLA haplotype of animals with the study epitopes will be required to evaluate the degree of immune protection conferred by epitope-driven DNA vaccines in pigs. PMID:27411061

  16. SELECTION OF NEW EPITOPES FROM MONOVALENT DISPLAYED PHAGE OCTAPEPTIDE LIBRARY

    Institute of Scientific and Technical Information of China (English)

    李全喜; 王琰; 李竞; 王雅明; 徐建军; 王力民; 董志伟

    1998-01-01

    A library of 2×l07 random oetspaptides was constructed by use of phegemid-based monovaient phage display system. The randomly synthesized degenerated oilgodeoxyribonucleotides (oligos) were fused to the truncated gⅢ (p210-p408). Sequeraze analysis of 11 randomly chosen clones suggested that the degenerated inserts and its deduced amino acid (an) sequences are randomly distributed. The library was used to select binding paptides to the morroeloncl antlhody (mAb) 9E10, which recognizes a continuous decapaptide epitope of denatured human c-myc protein. After four to five rounds of panning, most of the eluted clones could bind to 9E10. Sequerlce analysis of the selected positive clones indlcated that the binding sequences could fall into two chsses, one class (clone 1) shares a consensus motif, ISE x x L, with c-mire decapeprider and the sequences of the other class are entirely different. The binding of both classes to 9E10 could be specifically lnhlhited by froe c-myc deeapeptide. The immunogenlcitF cff the phage peptide was further investigsted h5, construction of multivalent displayed phage peptides and immunization of animals with or without adjuvant. ELISA and competitive ELISA showed that anti-serum from both mice and rabbit immunized with either done could bind to the original antigen, c-myc decapeptide. These results denote that in spite of the dissimilarity of the selected psptides with c-myc decapeptide, they are capable of inducing similar immune respones in vivo, thus actually mimicking the antigen epitope.

  17. Phase variation and conservation of lipooligosaccharide epitopes in Haemophilus somnus.

    Science.gov (United States)

    Inzana, T J; Hensley, J; McQuiston, J; Lesse, A J; Campagnari, A A; Boyle, S M; Apicella, M A

    1997-11-01

    The bovine-specific pathogen Haemophilus somnus is capable of undergoing structural and antigenic phase variation in its lipooligosaccharide (LOS) components after in vivo and in vitro passage. However, commensal isolates from the reproductive tract have not been observed to vary in phase (T. J. Inzana, R. P. Gogolewski, and L. B. Corbeil, Infect. Immun. 60:2943-2951, 1992). We now report that specific monoclonal antibodies (MAbs) to the LOSs of Haemophilus aegyptius, Neisseria gonorrhoeae, and Haemophilus influenzae, as well as H. somnus, reacted with some phase-variable epitopes in H. somnus LOS. All reactive MAbs bound to LOS components of about 4.3 kDa in the same H. somnus isolates, including a non-phase-varying strain. Following in vitro passage of a clonal variant of strain 738 that was nonreactive with the MAbs, 11.8% of young colonies shifted to a reactive phenotype. A digoxigenin-labelled 5'-CAATCAATCAATCAATCAATCAATCAAT-3' oligonucleotide probe hybridized to genomic DNA from strain 738 but did not react with DNA from a non-phase-varying strain. Sequence analysis of the gene containing 5'-CAAT-3' tandem sequences revealed 48% amino acid homology with the lex-2B gene-encoded protein of H. influenzae type b. Our results indicate that some LOS epitopes are conserved between H. somnus and other Haemophilus and Neisseria species, that LOS phase variation may occur at a high rate in some strains of H. somnus, and that phase variation may, in part, be due to 5'-CAAT-3' tandem sequences present in H. somnus genes.

  18. Immunoproteasome LMP2 60HH variant alters MBP epitope generation and reduces the risk to develop multiple sclerosis in Italian female population.

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    Michele Mishto

    Full Text Available BACKGROUND: Albeit several studies pointed out the pivotal role that CD4+T cells have in Multiple Sclerosis, the CD8+ T cells involvement in the pathology is still in its early phases of investigation. Proteasome degradation is the key step in the production of MHC class I-restricted epitopes and therefore its activity could be an important element in the activation and regulation of autoreactive CD8+ T cells in Multiple Sclerosis. METHODOLOGY/PRINCIPAL FINDINGS: Immunoproteasomes and PA28-alphabeta regulator are present in MS affected brain area and accumulated in plaques. They are expressed in cell types supposed to be involved in MS development such as neurons, endothelial cells, oligodendrocytes, macrophages/macroglia and lymphocytes. Furthermore, in a genetic study on 1262 Italian MS cases and 845 controls we observed that HLA-A*02+ female subjects carrying the immunoproteasome LMP2 codon 60HH variant have a reduced risk to develop MS. Accordingly, immunoproteasomes carrying the LMP2 60H allele produce in vitro a lower amount of the HLA-A*0201 restricted immunodominant epitope MBP(111-119. CONCLUSION/SIGNIFICANCE: The immunoproteasome LMP2 60HH variant reduces the risk to develop MS amongst Italian HLA-A*02+ females. We propose that such an effect is mediated by the altered proteasome-dependent production of a specific MBP epitope presented on the MHC class I. Our observations thereby support the hypothesis of an involvement of immunoproteasome in the MS pathogenesis.

  19. Computational Prediction and Identification of Epstein-Barr Virus Latent Membrane Protein 2A Antigen-Specific CD8+ T-Cell Epitopes

    Institute of Scientific and Technical Information of China (English)

    Bing Wang; Kun Yao; Genyan Liu; Fangyi Xie; Feng Zhou; Yun Chen

    2009-01-01

    Epstein-Barr virus (EBV) associated nasopharyngeal carcinoma (NPC) is a high incidence tumor in Southeast Asia. Among EBV encoded proteins, latent membrane protein 2A (LMP2A) is an important antigen for T cell therapy of EBV. In this study, we predicted six HLA-A2 restricted CTL candidate epitopes of LMP2A by SYFPEITHI, NetMHC and MHCPred methods combined with the polynomial method. Subsequently, biological functions of these peptides were tested by experiments in vitro. In ELISPOT assay, the positive response of the LMP2A specific CTL stimulated by three (LMP2A264-272, LMP2A426-434 and LMP2A356-364) of six peptides respectively showed that the numbers of spots forming cells (SFC) ranged from 55.7 to 80.6 SFC/5 × 104 CD8+ T cells and the responding index (RI) ranged from 5.4 to 7. These three epitope-specific CTLs could effectively kill specific HLA-A2-expressing target cells. As a result, LMP2A264-272 (QLSPLLGAV), LMP2A426-434 (CLGGLLTMV) and LMP2A356-364 (FLYALALLL) were identified as LMP2A-specific CD8+ T-cell epitopes. It would be useful to clarify immune response toward EBV and to develop a vaccine against EBV-correlative NPC. Cellular & Molecular Immunology.

  20. Induction of protective T-helper 1 immune responses against Echinococcus granulosus in mice by a multi-T-cell epitope antigen based on five proteins

    Directory of Open Access Journals (Sweden)

    Majid Esmaelizad

    2013-06-01

    Full Text Available In this study, we designed an experiment to predict a potential immunodominant T-cell epitope and evaluate the protectivity of this antigen in immunised mice. The T-cell epitopes of the candidate proteins (EgGST, EgA31, Eg95, EgTrp and P14-3-3 were detected using available web-based databases. The synthesised DNA was subcloned into the pET41a+ vector and expressed in Escherichia coli as a fusion to glutathione-S-transferase protein (GST. The resulting chimeric protein was then purified by affinity chromatography. Twenty female C57BL/6 mice were immunised with the antigen emulsified in Freund's adjuvant. Mouse splenocytes were then cultured in Dulbecco's Modified Eagle's Medium in the presence of the antigen. The production of interferon-γ was significantly higher in the immunised mice than in the control mice (> 1,300 pg/mL, but interleukin (IL-10 and IL-4 production was not statistically different between the two groups. In a challenge study in which mice were infected with 500 live protoscolices, a high protectivity level (99.6% was demonstrated in immunised BALB/C mice compared to the findings in the control groups [GST and adjuvant (Adj ]. These results demonstrate the successful application of the predicted T-cell epitope in designing a vaccine against Echinococcus granulosus in a mouse model.

  1. Low frequency of broadly neutralizing HIV antibodies during chronic infection even in quaternary epitope targeting antibodies containing large numbers of somatic mutations.

    Science.gov (United States)

    Hicar, Mark D; Chen, Xuemin; Kalams, Spyros A; Sojar, Hakimuddin; Landucci, Gary; Forthal, Donald N; Spearman, Paul; Crowe, James E

    2016-02-01

    Neutralizing antibodies (Abs) are thought to be a critical component of an appropriate HIV vaccine response. It has been proposed that Abs recognizing conformationally dependent quaternary epitopes on the HIV envelope (Env) trimer may be necessary to neutralize diverse HIV strains. A number of recently described broadly neutralizing monoclonal Abs (mAbs) recognize complex and quaternary epitopes. Generally, many such Abs exhibit extensive numbers of somatic mutations and unique structural characteristics. We sought to characterize the native antibody (Ab) response against circulating HIV focusing on such conformational responses, without a prior selection based on neutralization. Using a capture system based on VLPs incorporating cleaved envelope protein, we identified a selection of B cells that produce quaternary epitope targeting Abs (QtAbs). Similar to a number of broadly neutralizing Abs, the Ab genes encoding these QtAbs showed extensive numbers of somatic mutations. However, when expressed as recombinant molecules, these Abs failed to neutralize virus or mediate ADCVI activity. Molecular analysis showed unusually high numbers of mutations in the Ab heavy chain framework 3 region of the variable genes. The analysis suggests that large numbers of somatic mutations occur in Ab genes encoding HIV Abs in chronically infected individuals in a non-directed, stochastic, manner.

  2. Immunogenicity of porcine P[6], P[7]-specific △VP8* rotavirus subunit vaccines with a tetanus toxoid universal T cell epitope.

    Science.gov (United States)

    Wen, Xiaobo; Wei, Xiaoman; Ran, Xuhua; Ni, Hongbo; Cao, Si; Zhang, Yao

    2015-08-26

    Currently, commercial porcine rotavirus vaccines remain varied limitations. The objective of this study is to develop an alternative porcine rotavirus subunit vaccine candidate by parenteral administration, which enables to elicit robust immune responses against most prevalence porcine rotavirus strains. The bacterially-expressed porcine rotavirus P[6]- or P[7]-specific truncated VP8* (aa 64-223) recombinant protein with or without a universal tetanus toxoid CD4(+) T cell epitope P2 was generated. All the recombinant subunit proteins △VP8*s or P2-△VP8*s were of high solubility and high yields. The immunogenicity of each purified △VP8* and P2-△VP8* was evaluated in mice (10 μg/dose) or guinea pigs (20 μg/dose) immunized IM with 600 μg aluminum hydroxide three times at 2-week interval. The introduction of P2T cell epitope to P[7]-△VP8* elicited significantly higher IgG titer in mice than its absence. Comparatively, P2 epitope slightly enhanced the immunogenicity of P[6]-△VP8*. P2-P[7]△VP8* elicited high titer of neutralizing antibody against heterotypic P[7]-specific rotaviruses with varied G type combination. Our data indicated that two subunit vaccines could be plausible bivalent rotavirus vaccine candidate to provide antigenic coverage of porcine rotavirus strains of global or regional importance.

  3. Vaccination for 2009 pandemic H1N1 influenza A did not induce conserved epitope-specific memory CD8 T cell responses in HIV+ northern Thai children.

    Science.gov (United States)

    Chawansuntati, Kriangkrai; Aurpibul, Linda; Wipasa, Jiraprapa

    2015-09-11

    The influenza virus causes severe illness in susceptible populations, including children and people living with human immunodeficiency virus (HIV). Here, we investigated cell-mediated immune responses (CMI) against influenza CD8 T cell conserved epitopes in HIV-infected (HIV+) northern Thai children following the 2009 pandemic H1N1 influenza A vaccination. Sixty HIV+ children were vaccinated with two doses of the 2009 pandemic influenza vaccine and their CD8T cell responses were assessed. We found no significant differences in the increase of cytokines-producing and CD107a-expressing CD8+ T cells or CD8+ memory T cells in response to pooled conserved epitopes stimulation in vitro between children with different serologic responses to the vaccine at all time points of the study. Our results suggest that the 2009 pandemic H1N1 vaccine did not induce the conserved epitope-specific immune responses in HIV+ children. Vaccine design and vaccination strategy against influenza in these populations warrant further studies.

  4. T-cell recognition is shaped by epitope sequence conservation in the host proteome and microbiome

    DEFF Research Database (Denmark)

    Bresciani, Anne Gøther; Paul, Sinu; Schommer, Nina

    2016-01-01

    or allergen with the conservation of its sequence in the human proteome or the healthy human microbiome. Indeed, performing such comparisons on large sets of validated T-cell epitopes, we found that epitopes that are similar with self-antigens above a certain threshold showed lower immunogenicity, presumably...... as a result of negative selection of T cells capable of recognizing such peptides. Moreover, we also found a reduced level of immune recognition for epitopes conserved in the commensal microbiome, presumably as a result of peripheral tolerance. These findings indicate that the existence (and potentially...

  5. GAD65 epitope mapping and search for novel autoantibodies in GAD-associated neurological disorders.

    Science.gov (United States)

    Fouka, P; Alexopoulos, H; Akrivou, S; Trohatou, O; Politis, P K; Dalakas, M C

    2015-04-15

    Antibodies against Glutamic-acid-decarboxylase (GAD65) are seen in various CNS excitability disorders including stiff-person syndrome, cerebellar ataxia, encephalitis and epilepsy. To explore pathogenicity, we examined whether distinct epitope specificities or other co-existing antibodies may account for each disorder. The epitope recognized by all 27 tested patients, irrespective of clinical phenotype, corresponded to the catalytic core of GAD. No autoantibodies against known GABAergic antigens were found. In a screen for novel specificities using live hippocampal neurons, three epilepsy patients, but no other, were positive. We conclude that no GAD-specific epitope defines any neurological syndrome but other antibody specificities may account for certain phenotypes.

  6. CD4+ T cells targeting dominant and cryptic epitopes from Bacillus anthracis Lethal Factor

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    Stephanie eAscough

    2016-01-01

    Full Text Available Anthrax is an endemic infection in many countries, particularly in the developing world. The causative agent, Bacillus anthracis, mediates disease through the secretion of binary exotoxins. Until recently, research into adaptive immunity targeting this bacterial pathogen has largely focused on the humoral response to these toxins. There is, however, growing recognition that cellular immune responses involving IFNγ producing CD4+ T cells also contribute significantly to a protective memory response. An established concept in adaptive immunity to infection is that during infection of host cells, new microbial epitopes may be revealed, leading to immune recognition of so called ‘cryptic’ or ‘subdominant’ epitopes. We analysed the response to both cryptic and immunodominant T cell epitopes derived from the toxin component lethal factor and presented by a range of HLA-DR alleles. Using IFNγ-ELISPOT assays we characterised epitopes that elicited a response following immunisation with synthetic peptide and the whole protein and tested their capacities to bind purified HLA-DR molecules in vitro. We found that DR1 transgenics demonstrated T cell responses to a greater number of domain III cryptic epitopes than other HLA-DR transgenics, and that this pattern was repeated with the immunodominant epitopes, a greater proportion of these epitopes induced a T cell response when presented within the context of the whole protein. Immunodominant epitopes LF457-476 and LF467-487 were found to induce a T cell response to the peptide, as well as to the whole native LF protein in DR1 and DR15, but not in DR4 trangenics. The analysis of Domain I revealed the presence of several unique cryptic epitopes all of which showed a strong to moderate relative binding affinity to HLA-DR4 molecules. However, none of the cryptic epitopes from either domain III or I displayed notably high binding affinities across all HLA-DR alleles assayed. These responses were

  7. Large-scale validation of methods for cytotoxic T-lymphocyte epitope prediction

    DEFF Research Database (Denmark)

    Larsen, Mette Voldby; Lundegaard, Claus; Lamberth, K.;

    2007-01-01

    BACKGROUND: Reliable predictions of Cytotoxic T lymphocyte (CTL) epitopes are essential for rational vaccine design. Most importantly, they can minimize the experimental effort needed to identify epitopes. NetCTL is a web-based tool designed for predicting human CTL epitopes in any given protein....... of the other methods achieved a sensitivity of 0.64. The NetCTL-1.2 method is available at http://www.cbs.dtu.dk/services/NetCTL.All used datasets are available at http://www.cbs.dtu.dk/suppl/immunology/CTL-1.2.php....

  8. Discovery of novel targets for multi-epitope vaccines: Screening of HIV-1 genomes using association rule mining

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    Piontkivska Helen

    2009-07-01

    Full Text Available Abstract Background Studies have shown that in the genome of human immunodeficiency virus (HIV-1 regions responsible for interactions with the host's immune system, namely, cytotoxic T-lymphocyte (CTL epitopes tend to cluster together in relatively conserved regions. On the other hand, "epitope-less" regions or regions with relatively low density of epitopes tend to be more variable. However, very little is known about relationships among epitopes from different genes, in other words, whether particular epitopes from different genes would occur together in the same viral genome. To identify CTL epitopes in different genes that co-occur in HIV genomes, association rule mining was used. Results Using a set of 189 best-defined HIV-1 CTL/CD8+ epitopes from 9 different protein-coding genes, as described by Frahm, Linde & Brander (2007, we examined the complete genomic sequences of 62 reference HIV sequences (including 13 subtypes and sub-subtypes with approximately 4 representative sequences for each subtype or sub-subtype, and 18 circulating recombinant forms. The results showed that despite inclusion of recombinant sequences that would be expected to break-up associations of epitopes in different genes when two different genomes are recombined, there exist particular combinations of epitopes (epitope associations that occur repeatedly across the world-wide population of HIV-1. For example, Pol epitope LFLDGIDKA is found to be significantly associated with epitopes GHQAAMQML and FLKEKGGL from Gag and Nef, respectively, and this association rule is observed even among circulating recombinant forms. Conclusion We have identified CTL epitope combinations co-occurring in HIV-1 genomes including different subtypes and recombinant forms. Such co-occurrence has important implications for design of complex vaccines (multi-epitope vaccines and/or drugs that would target multiple HIV-1 regions at once and, thus, may be expected to overcome challenges

  9. Epitope-based vaccines with the Anaplasma marginale MSP1a functional motif induce a balanced humoral and cellular immune response in mice.

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    Paula S Santos

    Full Text Available Bovine anaplasmosis is a hemoparasitic disease that causes considerable economic loss to the dairy and beef industries. Cattle immunized with the Anaplasma marginale MSP1 outer membrane protein complex presents a protective humoral immune response; however, its efficacy is variable. Immunodominant epitopes seem to be a key-limiting factor for the adaptive immunity. We have successfully demonstrated that critical motifs of the MSP1a functional epitope are essential for antibody recognition of infected animal sera, but its protective immunity is yet to be tested. We have evaluated two synthetic vaccine formulations against A. marginale, using epitope-based approach in mice. Mice infection with bovine anaplasmosis was demonstrated by qPCR analysis of erythrocytes after 15-day exposure. A proof-of-concept was obtained in this murine model, in which peptides conjugated to bovine serum albumin were used for immunization in three 15-day intervals by intraperitoneal injections before challenging with live bacteria. Blood samples were analyzed for the presence of specific IgG2a and IgG1 antibodies, as well as for the rickettsemia analysis. A panel containing the cytokines' transcriptional profile for innate and adaptive immune responses was carried out through qPCR. Immunized BALB/c mice challenged with A. marginale presented stable body weight, reduced number of infected erythrocytes, and no mortality; and among control groups mortality rates ranged from 15% to 29%. Additionally, vaccines have significantly induced higher IgG2a than IgG1 response, followed by increased expression of pro-inflammatory cytokines. This is a successful demonstration of epitope-based vaccines, and protection against anaplasmosis may be associated with elicitation of effector functions of humoral and cellular immune responses in murine model.

  10. Generation, characterization and epitope mapping of two neutralizing and protective human recombinant antibodies against influenza A H5N1 viruses.

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    Lina Sun

    Full Text Available BACKGROUND: The development of new therapeutic targets and strategies to control highly pathogenic avian influenza (HPAI H5N1 virus infection in humans is urgently needed. Broadly cross-neutralizing recombinant human antibodies obtained from the survivors of H5N1 avian influenza provide an important role in immunotherapy for human H5N1 virus infection and definition of the critical epitopes for vaccine development. METHODOLOGY/PRINCIPAL FINDINGS: We have characterized two recombinant baculovirus-expressed human antibodies (rhAbs, AVFluIgG01 and AVFluIgG03, generated by screening a Fab antibody phage library derived from a patient recovered from infection with a highly pathogenic avian influenza A H5N1 clade 2.3 virus. AVFluIgG01 cross-neutralized the most of clade 0, clade 1, and clade 2 viruses tested, in contrast, AVFluIgG03 only neutralized clade 2 viruses. Passive immunization of mice with either AVFluIgG01 or AVFluIgG03 antibody resulted in protection from a lethal H5N1 clade 2.3 virus infection. Furthermore, through epitope mapping, we identify two distinct epitopes on H5 HA molecule recognized by these rhAbs and demonstrate their potential to protect against a lethal H5N1 virus infection in a mouse model. CONCLUSIONS/SIGNIFICANCE: Importantly, localization of the epitopes recognized by these two neutralizing and protective antibodies has provided, for the first time, insight into the human antibody responses to H5N1 viruses which contribute to the H5 immunity in the recovered patient. These results highlight the potential of a rhAbs treatment strategy for human H5N1 virus infection and provide new insight for the development of effective H5N1 pandemic vaccines.

  11. Epitope mapping and identification of amino acids critical for mouse IgG-binding to linear epitopes on Gly m Bd 28K.

    Science.gov (United States)

    Xi, Jun; Yan, Huili

    2016-10-01

    Gly m Bd 28K is one of the major allergens in soybeans, but there is limited information on its IgG-binding epitopes. Thirty-four overlapping peptides that covered the entire sequence of Gly m Bd 28K were synthesized, and 3 monoclonal antibodies against Gly m Bd 28K were utilized to identify the IgG-binding regions of Gly m Bd 28K. Three dominant peptides corresponding to (28)GDKKSPKSLFLMSNS(42)(G28-S42), (56)LKSHGGRIFYRHMHI(70)(L56-I70), and (154)ETFQSFYIGGGANSH(168)(E154-H168) were recognized. L56-I70 is the most important epitope, and a competitive ELISA indicated that it could inhibit the binding of monoclonal antibody to Gly m Bd 28K protein. Alanine scanning of L56-I70 documented that F64, Y65, and R66 were the critical amino acids of this epitope. Two bioinformatics tools, ABCpred and BepiPred, were used to predict the epitopes of Gly m Bd 28K, and the predictions were compared with the epitopes that we had located by monoclonal antibodies.

  12. T Cell Epitope Immunotherapy Induces a CD4+ T Cell Population with Regulatory Activity

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    Verhoef Adrienne

    2005-01-01

    Full Text Available Background Synthetic peptides, representing CD4+ T cell epitopes, derived from the primary sequence of allergen molecules have been used to down-regulate allergic inflammation in sensitised individuals. Treatment of allergic diseases with peptides may offer substantial advantages over treatment with native allergen molecules because of the reduced potential for cross-linking IgE bound to the surface of mast cells and basophils. Methods and Findings In this study we address the mechanism of action of peptide immunotherapy (PIT in cat-allergic, asthmatic patients. Cell-division-tracking dyes, cell-mixing experiments, surface phenotyping, and cytokine measurements were used to investigate immunomodulation in peripheral blood mononuclear cells (PBMCs after therapy. Proliferative responses of PBMCs to allergen extract were significantly reduced after PIT. This was associated with modified cytokine profiles generally characterised by an increase in interleukin-10 and a decrease in interleukin-5 production. CD4+ cells isolated after PIT were able to actively suppress allergen-specific proliferative responses of pretreatment CD4neg PBMCs in co-culture experiments. PIT was associated with a significant increase in surface expression of CD5 on both CD4+ and CD8+ PBMCs. Conclusion This study provides evidence for the induction of a population of CD4+ T cells with suppressor/regulatory activity following PIT. Furthermore, up-regulation of cell surface levels of CD5 may contribute to reduced reactivity to allergen.

  13. Analysis of Conformational B-Cell Epitopes in the Antibody-Antigen Complex Using the Depth Function and the Convex Hull.

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    Wei Zheng

    Full Text Available The prediction of conformational b-cell epitopes plays an important role in immunoinformatics. Several computational methods are proposed on the basis of discrimination determined by the solvent-accessible surface between epitopes and non-epitopes, but the performance of existing methods is far from satisfying. In this paper, depth functions and the k-th surface convex hull are used to analyze epitopes and exposed non-epitopes. On each layer of the protein, we compute relative solvent accessibility and four different types of depth functions, i.e., Chakravarty depth, DPX, half-sphere exposure and half space depth, to analyze the location of epitopes on different layers of the proteins. We found that conformational b-cell epitopes are rich in charged residues Asp, Glu, Lys, Arg, His; aliphatic residues Gly, Pro; non-charged residues Asn, Gln; and aromatic residue Tyr. Conformational b-cell epitopes are rich in coils. Conservation of epitopes is not significantly lower than that of exposed non-epitopes. The average depths (obtained by four methods for epitopes are significantly lower than that of non-epitopes on the surface using the Wilcoxon rank sum test. Epitopes are more likely to be located in the outer layer of the convex hull of a protein. On the benchmark dataset, the cumulate 10th convex hull covers 84.6% of exposed residues on the protein surface area, and nearly 95% of epitope sites. These findings may be helpful in building a predictor for epitopes.

  14. An overview of bioinformatics tools for epitope prediction: implications on vaccine development.

    Science.gov (United States)

    Soria-Guerra, Ruth E; Nieto-Gomez, Ricardo; Govea-Alonso, Dania O; Rosales-Mendoza, Sergio

    2015-02-01

    Exploitation of recombinant DNA and sequencing technologies has led to a new concept in vaccination in which isolated epitopes, capable of stimulating a specific immune response, have been identified and used to achieve advanced vaccine formulations; replacing those constituted by whole pathogen-formulations. In this context, bioinformatics approaches play a critical role on analyzing multiple genomes to select the protective epitopes in silico. It is conceived that cocktails of defined epitopes or chimeric protein arrangements, including the target epitopes, may provide a rationale design capable to elicit convenient humoral or cellular immune responses. This review presents a comprehensive compilation of the most advantageous online immunological software and searchable, in order to facilitate the design and development of vaccines. An outlook on how these tools are supporting vaccine development is presented. HIV and influenza have been taken as examples of promising developments on vaccination against hypervariable viruses. Perspectives in this field are also envisioned.

  15. Characterization of epitopes recognized by monoclonal antibodies: experimental approaches supported by freely accessible bioinformatic tools.

    Science.gov (United States)

    Clementi, Nicola; Mancini, Nicasio; Castelli, Matteo; Clementi, Massimo; Burioni, Roberto

    2013-05-01

    Monoclonal antibodies (mAbs) have been used successfully both in research and for clinical purposes. The possible use of protective mAbs directed against different microbial pathogens is currently being considered. The fine definition of the epitope recognized by a protective mAb is an important aspect to be considered for possible development in epitope-based vaccinology. The most accurate approach to this is the X-ray resolution of mAb/antigen crystal complex. Unfortunately, this approach is not always feasible. Under this perspective, several surrogate epitope mapping strategies based on the use of bioinformatics have been developed. In this article, we review the most common, freely accessible, bioinformatic tools used for epitope characterization and provide some basic examples of molecular visualization, editing and computational analysis.

  16. Rotavirus VP7 epitope chimeric proteins elicit cross-immunoreactivity in guinea pigs

    Institute of Scientific and Technical Information of China (English)

    Bingxin; Zhao; Xiaoxia; Pan; Yumei; Teng; Wenyue; Xia; Jing; Wang; Yuling; Wen; Yuanding; Chen

    2015-01-01

    VP7 of group A rotavirus(RVA) contains major neutralizing epitopes. Using the antigenic protein VP6 as the vector, chimeric proteins carrying foreign epitopes have been shown to possess good immunoreactivity and immunogenicity. In the present study, using modified VP6 as the vector,three chimeric proteins carrying epitopes derived from VP7 of RVA were constructed. The results showed that the chimeric proteins reacted with anti-VP6 and with SA11 and Wa virus strains.Antibodies from guinea pigs inoculated with the chimeric proteins recognized VP6 and VP7 of RVA and protected mammalian cells from SA11 and Wa infection in vitro. The neutralizing activities of the antibodies against the chimeric proteins were significantly higher than those against the vector protein VP6 F. Thus, development of chimeric vaccines carrying VP7 epitopes using VP6 as a vector could be a promising alternative to enhance immunization against RVAs.

  17. Analysis of epitopes in the capsid protein of avian hepatitis E virus by using monoclonal antibodies.

    Science.gov (United States)

    Dong, Shiwei; Zhao, Qin; Lu, Mingzhe; Sun, Peiming; Qiu, Hongkai; Zhang, Lu; Lv, Junhua; Zhou, En-Min

    2011-02-01

    Avian hepatitis E virus (HEV) is related genetically and antigenically to human and swine HEVs and capsid protein of avian HEV shares approximately 48-49% amino acid sequence identities with those of human and swine HEVs. Six monoclonal antibodies (MAbs) were produced and used to locate different epitopes in the ORF2 region of aa 339-570 of avian HEV Chinese isolate. The results showed that five epitopes were located in the aa 339-414 region and one in the aa 510-515 region. Two epitopes located in aa 339-355 and aa 384-414 regions are the immunodominant epitopes on the surface of the avian HEV particles as demonstrated by immune capture of viral particles and immunohistochemical detection of the ORF2 antigens with two MAbs.

  18. Longitudinal epitope mapping in MuSK myasthenia gravis: implications for disease severity.

    Science.gov (United States)

    Huijbers, Maartje G; Vink, Anna-Fleur D; Niks, Erik H; Westhuis, Ruben H; van Zwet, Erik W; de Meel, Robert H; Rojas-García, Ricardo; Díaz-Manera, Jordi; Kuks, Jan B; Klooster, Rinse; Straasheijm, Kirsten; Evoli, Amelia; Illa, Isabel; van der Maarel, Silvère M; Verschuuren, Jan J

    2016-02-15

    Muscle weakness in MuSK myasthenia gravis (MG) is caused predominantly by IgG4 antibodies which block MuSK signalling and destabilize neuromuscular junctions. We determined whether the binding pattern of MuSK IgG4 antibodies change throughout the disease course ("epitope spreading"), and affect disease severity or treatment responsiveness. We mapped the MuSK epitopes of 255 longitudinal serum samples of 53 unique MuSK MG patients from three independent cohorts with ELISA. Antibodies against the MuSK Iglike-1 domain determine disease severity. Epitope spreading outside this domain did not contribute to disease severity nor to pyridostigmine responsiveness. This provides a rationale for epitope specific treatment strategies.

  19. High-resolution mapping of linear antibody epitopes using ultrahigh-density peptide microarrays

    DEFF Research Database (Denmark)

    Buus, Søren; Rockberg, Johan; Forsström, Björn

    2012-01-01

    -resolution mapping of linear antibody epitopes, we have used ultrahigh-density peptide microarrays generating several hundred thousand different peptides per array. Using exhaustive length and substitution analysis, we have successfully examined the specificity of a panel of polyclonal antibodies raised against......Antibodies empower numerous important scientific, clinical, diagnostic, and industrial applications. Ideally, the epitope(s) targeted by an antibody should be identified and characterized, thereby establishing antibody reactivity, highlighting possible cross-reactivities, and perhaps even warning...... against unwanted (e.g. autoimmune) reactivities. Antibodies target proteins as either conformational or linear epitopes. The latter are typically probed with peptides, but the cost of peptide screening programs tends to prohibit comprehensive specificity analysis. To perform high-throughput, high...

  20. Rotavirus VP7 epitope chimeric proteins elicit cross-immunoreactivity in guinea pigs.

    Science.gov (United States)

    Zhao, Bingxin; Pan, Xiaoxia; Teng, Yumei; Xia, Wenyue; Wang, Jing; Wen, Yuling; Chen, Yuanding

    2015-10-01

    VP7 of group A rotavirus (RVA) contains major neutralizing epitopes. Using the antigenic protein VP6 as the vector, chimeric proteins carrying foreign epitopes have been shown to possess good immunoreactivity and immunogenicity. In the present study, using modified VP6 as the vector, three chimeric proteins carrying epitopes derived from VP7 of RVA were constructed. The results showed that the chimeric proteins reacted with anti-VP6 and with SA11 and Wa virus strains. Antibodies from guinea pigs inoculated with the chimeric proteins recognized VP6 and VP7 of RVA and protected mammalian cells from SA11 and Wa infection in vitro. The neutralizing activities of the antibodies against the chimeric proteins were significantly higher than those against the vector protein VP6F. Thus, development of chimeric vaccines carrying VP7 epitopes using VP6 as a vector could be a promising alternative to enhance immunization against RVAs.

  1. Delineation and comparison of ganglioside-binding epitopes for the toxins of Vibrio cholerae, Escherichia coli, and Clostridium tetani: evidence for overlapping epitopes.

    Science.gov (United States)

    Angström, J; Teneberg, S; Karlsson, K A

    1994-12-06

    Binding studies of various glycolipids, mainly belonging to the ganglio series, to the toxins isolated from Vibrio cholerae, Escherichia coli, and Clostridium tetani have been performed, using the microtiter well assay. By using the found binding preferences in conjunction with minimum-energy conformations obtained from molecular modeling of the various ligands, binding epitopes on the natural receptor glycolipids for the toxins have been defined. The binding preferences for the cholera toxin and the heat-labile E. coli toxin are very similar, with the ganglioside GM1 being the most efficient ligand. The tetanus toxin binds strongly to gangliosides of the G1b series, with GT1b as the most efficient ligand. It is found that the binding epitope on GM1 for the cholera and heat-labile toxins to a large extent overlaps with the epitope on GQ1b for the tetanus toxin.

  2. Construction and immunogenicity prediction of Plasmodium falciparum CTL epitope minigene vaccine

    Institute of Scientific and Technical Information of China (English)

    TANG; Yuyang

    2001-01-01

    the peptide-MHC class I complex limits the binding rate of exogenous peptide, J. Immunol., 1993, 151: 6020.[13] Lin, C. T., Jiang, Y. F., Yin, B. et al., The application of isocaudamers cloning technique in construction of Plasmodium falciparum multi-valent vaccine, J. Biochem. & Biophys. (in Chinese), 1999, 15: 973.[14] Goligan, L. I., Kruisbeek, A. M., Margulies, D. H. et al., Current Protocols in Immunology, New York: Greene Publishing Associates and Wiley-Interscience, 1991, 9.0.1-9.4.11.[15] Zemmour, J., Little, A. M., Schendel, D. et al., The HLA-A, B, C "negative" mutant cell line C1R expresses a novel HLA-B35 allele, which also has a point mutations in the translation initiation codon, J. Immunol., 1992, 148: 1941.[16] Van der Burg, S. H., Visseren, M. J. W., Brandt, R. M. P. et al., Immunogenicity of peptides bound to MHC class I molecules depends on the MHC-peptide complex stability, J. Immunol.,1996, 156: 3308.[17] Barnstable, C. J., Bodmer, W. F., Brown, G. et al., Production of monoclonal antibodies to group A erythrocytes, HLA and other human cell surface antigens--new tools for genetic analysis, Cell, 1978, 14(1): 9.[18] Aidoo, M., Lalvani, A., Allsopp, C. E. M. et al., Identification of conserved antigenic components for a cytotoxic T lymphocyte-inducing vaccine against malaria, Lancet, 1995, 345: 1003.[19] Feltkamp, M. C. W., Vierboom, M. P. M., Kast, W. M. et al., Efficient MHC class I-peptide binding is required but does not ensure MHC class I-restricted immunogenicity, Mol. Immunol., 1994, 31: 1391.[20] Sette, A., Vitiello, A., Reherman, B. et al., The relationship between class I binding affinity and immunogenicity of potential cytotoxic T cell epitopes, J. Immunol., 1994, 153: 5586.[21] Del, V. M. D., Schlicht, H. J., Ruppert, T. et al., Efficient processing of an antigenic sequence for presentation by MHC class I molecules depends on its neighboring residues in the protein, Cell, 1990, 66: 1145.[22

  3. Analysis of potato virus Y coat protein epitopes recognized by three commercial monoclonal antibodies.

    Directory of Open Access Journals (Sweden)

    Yan-Ping Tian

    Full Text Available BACKGROUND: Potato virus Y (PVY, genus Potyvirus causes substantial economic losses in solanaceous plants. Routine screening for PVY is an essential part of seed potato certification, and serological assays are often used. The commercial, commonly used monoclonal antibodies, MAb1128, MAb1129, and MAb1130, recognize the viral coat protein (CP of PVY and distinguish PVYN strains from PVYO and PVYC strains, or detect all PVY strains, respectively. However, the minimal epitopes recognized by these antibodies have not been identified. METHODOLOGY/PRINCIPAL FINDINGS: SPOT peptide array was used to map the epitopes in CP recognized by MAb1128, MAb1129, and MAb1130. Then alanine replacement as well as N- and C-terminal deletion analysis of the identified peptide epitopes was done to determine critical amino acids for antibody recognition and the respective minimal epitopes. The epitopes of all antibodies were located within the 30 N-terminal-most residues. The minimal epitope of MAb1128 was 25NLNKEK30. Replacement of 25N or 27N with alanine weakened the recognition by MAb1128, and replacement of 26L, 29E, or 30K nearly precluded recognition. The minimal epitope for MAb1129 was 16RPEQGSIQSNP26 and the most critical residues for recognition were 22I and 23Q. The epitope of MAb1130 was defined by residues 5IDAGGS10. Mutation of residue 6D abrogated and mutation of 9G strongly reduced recognition of the peptide by MAb1130. Amino acid sequence alignment demonstrated that these epitopes are relatively conserved among PVY strains. Finally, recombinant CPs were produced to demonstrate that mutations in the variable positions of the epitope regions can affect detection with the MAbs. CONCLUSIONS/SIGNIFICANCE: The epitope data acquired can be compared with data on PVY CP-encoding sequences produced by laboratories worldwide and utilized to monitor how widely the new variants of PVY can be detected with current seed potato certification schemes or during the

  4. High frequency of T cells specific for cryptic epitopes in melanoma patients

    Science.gov (United States)

    Andersen, Rikke Sick; Andersen, Sofie Ramskov; Hjortsø, Mads Duus; Lyngaa, Rikke; Idorn, Manja; Køllgård, Tania Maria; Met, Özcan; thor Straten, Per; Hadrup, Sine Reker

    2013-01-01

    A number of cytotoxic T-cell epitopes are cryptic epitopes generated from non-conventional sources. These include epitopes that are encoded by alternative open reading frames or in generally non-coding genomic regions, such as introns. We have previously observed a frequent recognition of cryptic epitopes by tumor infiltrating lymphocytes isolated from melanoma patients. Here, we show that such cryptic epitopes are more frequently recognized than antigens of the same class encoded by canonical reading frames. Furthermore, we report the presence of T cells specific for three cryptic epitopes encoded in intronic sequences, as a result of incomplete splicing, in the circulation of melanoma patients. One of these epitopes derives from antigen isolated from immunoselected melanoma 2 (AIM2), while the two others are encoded in an alternative open reading frame of an incompletely spliced form of N-acetylglucosaminyl-transferase V (GNT-V) known as NA17-A. We have detected frequent T-cell responses against AIM2 and NA17-A epitopes in the blood of melanoma patients, both prior and after one round of in vitro peptide stimulation, but not in the circulation of healthy individuals and patients with breast or renal carcinoma. In summary, our findings indicate that the T-cell reactivity against AIM2 and NA17-A in the blood of melanoma patients is extensive, suggesting that—similar to melan A (also known as MART1)—these antigens might be used for immunomonitoring or as model antigens in several clinical and preclinical settings. PMID:24073381

  5. Selective pressure to increase charge in immunodominant epitopes of the H3 hemagglutinin influenza protein.

    Science.gov (United States)

    Pan, Keyao; Long, Jinxue; Sun, Haoxin; Tobin, Gregory J; Nara, Peter L; Deem, Michael W

    2011-01-01

    The evolutionary speed and the consequent immune escape of H3N2 influenza A virus make it an interesting evolutionary system. Charged amino acid residues are often significant contributors to the free energy of binding for protein-protein interactions, including antibody-antigen binding and ligand-receptor binding. We used Markov chain theory and maximum likelihood estimation to model the evolution of the number of charged amino acids on the dominant epitope in the hemagglutinin protein of circulating H3N2 virus strains. The number of charged amino acids increased in the dominant epitope B of the H3N2 virus since introduction in humans in 1968. When epitope A became dominant in 1989, the number of charged amino acids increased in epitope A and decreased in epitope B. Interestingly, the number of charged residues in the dominant epitope of the dominant circulating strain is never fewer than that in the vaccine strain. We propose these results indicate selective pressure for charged amino acids that increase the affinity of the virus epitope for water and decrease the affinity for host antibodies. The standard PAM model of generic protein evolution is unable to capture these trends. The reduced alphabet Markov model (RAMM) model we introduce captures the increased selective pressure for charged amino acids in the dominant epitope of hemagglutinin of H3N2 influenza (R (2) > 0.98 between 1968 and 1988). The RAMM model calibrated to historical H3N2 influenza virus evolution in humans fit well to the H3N2/Wyoming virus evolution data from Guinea pig animal model studies.

  6. Screening and identification of novel B cell epitopes of Toxoplasma gondii SAG1

    OpenAIRE

    Wang, Yanhua; Wang, Guangxiang; Zhang, Delin; Yin, Hong; Wang, Meng

    2013-01-01

    Background The identification of protein epitopes is useful for diagnostic purposes and for the development of peptide vaccines. In this study, the epitopes of Toxoplasma gondii SAG1 were identified using synthetic peptide techniques with the aid of bioinformatics. Findings Eleven peptides derived from T. gondii SAG1 were assessed by ELISA using pig sera from different time points after infection. Four (PS4, PS6, PS10 and PS11), out of the eleven peptides tested were recognized by all sera. T...

  7. From viral genome to specific peptide epitopes: methods for identifying porcine T cell epitopes based on in silico predictions, in vitro identification and ex vivo verification

    DEFF Research Database (Denmark)

    Pedersen, Lasse Eggers; Rasmussen, Michael; Harndah, Mikkel;

    2013-01-01

    The affinity with which major histocompatibility complex (MHC) class I molecules bind peptides is instrumental to presentation of viral epitopes to cytotoxic T lymphocytes (CTLs). We analyzed three swine leukocyte antigen (SLA) molecules for complete nonamer peptide-based binding matrices in order.......000 peptides. T cell epitopes were identified using peptide-SLA complexes assembled into fluorescent tetramers to stain swine influenza specific CTLs derived from immunized animals and MHC-defined pigs vaccinated against foot-and-mouth disease virus. These results demonstrate the broad applicability of methods...

  8. B-cell epitope mapping for the design of vaccines and effective diagnostics

    Directory of Open Access Journals (Sweden)

    Tarek A. Ahmad

    2016-01-01

    Full Text Available The increasing resistance of many microbial strains to antibiotics, delayed laboratory results, and side effects of many chemotherapeutics has raised the need to search for sensitive diagnostics and new prophylactic strategies especially prevention by vaccination. Understanding the epitope/antibody interaction is the key to constructing potent vaccines and effective diagnostics. B-cell epitope mapping is a promising approach to identifying the main antigenic determinants of microorganisms, in special concern the discontinuous conformational ones. Epitope-based vaccines have remarkable privilege over the conventional ones since they are specific, able to avoid undesirable immune responses, generate long lasting immunity, and are reasonably cheaper. This up-to-date review discusses and compares the different physical, computational, and molecular methods that have been used in epitope mapping. The role of each method in the identification of potent epitopes in viruses, bacteria, fungi, parasites, as well as human diseases are tagged and documented. Simultaneously, frequent combinatorial methods are highlighted. The article aims to assist researchers to design the most suitable protocol for mapping their B-cell epitopes.

  9. Whole-exome sequencing predicted cancer epitope trees of 23 early cervical cancers in Chinese women.

    Science.gov (United States)

    Li, Xia; Huang, Hailiang; Guan, Yanfang; Gong, Yuhua; He, Cheng-Yi; Yi, Xin; Qi, Ming; Chen, Zhi-Ying

    2017-01-01

    Emerging evidence suggest that the heterogeneity of cancer limits the efficacy of immunotherapy. To search for optimal therapeutic targets for enhancing the efficacy, we used whole-exome sequencing data of 23 early cervical tumors from Chinese women to investigate the hierarchical structure of the somatic mutations and the neo-epitopes. The putative neo-epitopes were predicted based on the mutant peptides' strong binding with major histocompatibility complex class I molecules. We found that each tumor carried an average of 117 mutations and 61 putative neo-epitopes. Each patient displayed a unique phylogenetic tree in which almost all subclones harbored neo-epitopes, highlighting the importance of individual neo-epitope tree in determination of immunotherapeutic targets. The alterations in FBXW7 and PIK3CA, or other members of the significantly altered ubiquitin-mediated proteolysis and extracellular matrix receptor interaction related pathways, were proposed as the earliest changes triggering the malignant progression. The neo-epitopes involved in these pathways, and located at the top of the hierarchy tree, might become the optimal candidates for therapeutic targets, possessing the potential to mediate T-cell killing of the descendant cells. These findings expanded our understanding in early stage of cervical carcinogenesis and offered an important approach to assist optimizing the immunotherapeutic target selection.

  10. Design of a heterosubtypic epitope-based peptide vaccine fused with hemokinin-1 against influenza viruses

    Institute of Scientific and Technical Information of China (English)

    Shahla; Shahsavandi; Mohammad; Majid; Ebrahimi; Kaveh; Sadeghi; Homayoon; Mahravani

    2015-01-01

    Influenza viruses continue to emerge and re-emerge, posing new threats for public health. Control and treatment of influenza depends mainly on vaccination and chemoprophylaxis with approved antiviral drugs. Identification of specific epitopes derived from influenza viruses has significantly advanced the development of epitope-based vaccines. Here, we explore the idea of using HLA binding data to design an epitope-based vaccine that can elicit heterosubtypic T-cell responses against circulating H7N9, H5N1, and H9N2 subtypes. The hemokinin-1(HK-1) peptide sequence was used to induce immune responses against the influenza viruses. Five conserved high score cytotoxic T lymphocyte(CTL) epitopes restricted to HLA-A*0201-binding peptides within the hemagglutinin(HA) protein of the viruses were chosen, and two HA CTL/HK-1 chimera protein models designed. Using in silico analysis, which involves interferon epitope scanning, protein structure prediction, antigenic epitope determination, and model quality evaluation, chimeric proteins were designed. The applicability of one of these proteins as a heterosubtypic epitopebased vaccine candidate was analyzed.

  11. Broadly neutralizing epitopes in the Plasmodium vivax vaccine candidate Duffy Binding Protein.

    Science.gov (United States)

    Chen, Edwin; Salinas, Nichole D; Huang, Yining; Ntumngia, Francis; Plasencia, Manolo D; Gross, Michael L; Adams, John H; Tolia, Niraj Harish

    2016-05-31

    Plasmodium vivax Duffy Binding Protein (PvDBP) is the most promising vaccine candidate for P. vivax malaria. The polymorphic nature of PvDBP induces strain-specific immune responses, however, and the epitopes of broadly neutralizing antibodies are unknown. These features hamper the rational design of potent DBP-based vaccines and necessitate the identification of globally conserved epitopes. Using X-ray crystallography, small-angle X-ray scattering, hydrogen-deuterium exchange mass spectrometry, and mutational mapping, we have defined epitopes for three inhibitory mAbs (mAbs 2D10, 2H2, and 2C6) and one noninhibitory mAb (3D10) that engage DBP. These studies expand the currently known inhibitory epitope repertoire by establishing protective motifs in subdomain three outside the receptor-binding and dimerization residues of DBP, and introduce globally conserved protective targets. All of the epitopes are highly conserved among DBP alleles. The identification of broadly conserved epitopes of inhibitory antibodies provides critical motifs that should be retained in the next generation of potent vaccines for P. vivax malaria.

  12. Characterization of Immunodominant BK Polyomavirus 9mer Epitope T Cell Responses

    Science.gov (United States)

    Cioni, M.; Leboeuf, C.; Comoli, P.; Ginevri, F.

    2016-01-01

    Uncontrolled BK polyomavirus (BKPyV) replication in kidney transplant recipients (KTRs) causes polyomavirus‐associated nephropathy and allograft loss. Reducing immunosuppression is associated with clearing viremia and nephropathy and increasing BKPyV‐specific T cell responses in most patients; however, current immunoassays have limited sensitivity, target mostly CD4+ T cells, and largely fail to predict onset and clearance of BKPyV replication. To characterize BKPyV‐specific CD8+ T cells, bioinformatics were used to predict 9mer epitopes in the early viral gene region (EVGR) presented by 14 common HLAs in Europe and North America. Thirty‐nine EVGR epitopes were experimentally confirmed by interferon‐γ enzyme‐linked immunospot assays in at least 30% of BKPyV IgG–seropositive healthy participants. Most 9mers clustered in domains, and some were presented by more than one HLA class I, as typically seen for immunodominant epitopes. Specific T cell binding using MHC class I streptamers was demonstrated for 21 of 39 (54%) epitopes. In a prospective cohort of 118 pediatric KTRs, 19 patients protected or recovering from BKPyV viremia were experimentally tested, and 13 epitopes were validated. Single HLA mismatches were not associated with viremia, suggesting that failing immune control likely involves multiple factors including maintenance immunosuppression. Combining BKPyV load and T cell assays using immunodominant epitopes may help in evaluating risk and reducing immunosuppression and may lead to safe adoptive T cell transfer. PMID:26663765

  13. Topology of the C-terminal tail of HIV-1 gp41: differential exposure of the Kennedy epitope on cell and viral membranes.

    Science.gov (United States)

    Steckbeck, Jonathan D; Sun, Chengqun; Sturgeon, Timothy J; Montelaro, Ronald C

    2010-01-01

    The C-terminal tail (CTT) of the HIV-1 gp41 envelope (Env) protein is increasingly recognized as an important determinant of Env structure and functional properties, including fusogenicity and antigenicity. While the CTT has been commonly referred to as the "intracytoplasmic domain" based on the assumption of an exclusive localization inside the membrane lipid bilayer, early antigenicity studies and recent biochemical analyses have produced a credible case for surface exposure of specific CTT sequences, including the classical "Kennedy epitope" (KE) of gp41, leading to an alternative model of gp41 topology with multiple membrane-spanning domains. The current study was designed to test these conflicting models of CTT topology by characterizing the exposure of native CTT sequences and substituted VSV-G epitope tags in cell- and virion-associated Env to reference monoclonal antibodies (MAbs). Surface staining and FACS analysis of intact, Env-expressing cells demonstrated that the KE is accessible to binding by MAbs directed to both an inserted VSV-G epitope tag and the native KE sequence. Importantly, the VSV-G tag was only reactive when inserted into the KE; no reactivity was observed in cells expressing Env with the VSV-G tag inserted into the LLP2 domain. In contrast to cell-surface expressed Env, no binding of KE-directed MAbs was observed to Env on the surface of intact virions using either immune precipitation or surface plasmon resonance spectroscopy. These data indicate apparently distinct CTT topologies for virion- and cell-associated Env species and add to the case for a reconsideration of CTT topology that is more complex than currently envisioned.

  14. Topology of the C-terminal tail of HIV-1 gp41: differential exposure of the Kennedy epitope on cell and viral membranes.

    Directory of Open Access Journals (Sweden)

    Jonathan D Steckbeck

    Full Text Available The C-terminal tail (CTT of the HIV-1 gp41 envelope (Env protein is increasingly recognized as an important determinant of Env structure and functional properties, including fusogenicity and antigenicity. While the CTT has been commonly referred to as the "intracytoplasmic domain" based on the assumption of an exclusive localization inside the membrane lipid bilayer, early antigenicity studies and recent biochemical analyses have produced a credible case for surface exposure of specific CTT sequences, including the classical "Kennedy epitope" (KE of gp41, leading to an alternative model of gp41 topology with multiple membrane-spanning domains. The current study was designed to test these conflicting models of CTT topology by characterizing the exposure of native CTT sequences and substituted VSV-G epitope tags in cell- and virion-associated Env to reference monoclonal antibodies (MAbs. Surface staining and FACS analysis of intact, Env-expressing cells demonstrated that the KE is accessible to binding by MAbs directed to both an inserted VSV-G epitope tag and the native KE sequence. Importantly, the VSV-G tag was only reactive when inserted into the KE; no reactivity was observed in cells expressing Env with the VSV-G tag inserted into the LLP2 domain. In contrast to cell-surface expressed Env, no binding of KE-directed MAbs was observed to Env on the surface of intact virions using either immune precipitation or surface plasmon resonance spectroscopy. These data indicate apparently distinct CTT topologies for virion- and cell-associated Env species and add to the case for a reconsideration of CTT topology that is more complex than currently envisioned.

  15. The fully synthetic MAG-Tn3 therapeutic vaccine containing the tetanus toxoid-derived TT830-844 universal epitope provides anti-tumor immunity.

    Science.gov (United States)

    Laubreton, Daphné; Bay, Sylvie; Sedlik, Christine; Artaud, Cécile; Ganneau, Christelle; Dériaud, Edith; Viel, Sophie; Puaux, Anne-Laure; Amigorena, Sebastian; Gérard, Catherine; Lo-Man, Richard; Leclerc, Claude

    2016-03-01

    Malignant transformations are often associated with aberrant glycosylation processes that lead to the expression of new carbohydrate antigens at the surface of tumor cells. Of these carbohydrate antigens, the Tn antigen is particularly highly expressed in many carcinomas, especially in breast carcinoma. We designed MAG-Tn3, a fully synthetic vaccine based on three consecutive Tn moieties that are O-linked to a CD4+ T cell epitope, to induce anti-Tn antibody responses that could be helpful for therapeutic vaccination against cancer. To ensure broad coverage within the human population, the tetanus toxoid-derived peptide TT830-844 was selected as a T-helper epitope because it can bind to various HLA-DRB molecules. We showed that the MAG-Tn3 vaccine, which was formulated with the GSK proprietary immunostimulant AS15 and designed for human cancer therapy, is able to induce an anti-Tn antibody response in mice of various H-2 haplotypes, and this response correlates with the ability to induce a specific T cell response against the TT830-844 peptide. The universality of the TT830-844 peptide was extended to new H-2 and HLA-DRB molecules that were capable of binding this T cell epitope. Finally, the MAG-Tn3 vaccine was able to induce anti-Tn antibody responses in cynomolgus monkeys, which targeted Tn-expressing tumor cells and mediated tumor cell death both in vitro and in vivo. Thus, MAG-Tn3 is a highly promising anticancer vaccine that is currently under evaluation in a phase I clinical trial.

  16. Association of New Putative Epitopes of Myelin Proteolipid Protein (58-74 with Pathogenesis of Multiple Sclerosis

    Directory of Open Access Journals (Sweden)

    Zahra Zamanzadeh

    2016-10-01

    Full Text Available Multiple sclerosis (MS is an autoimmune disease in which auto-reactive T cells react with self-antigens expressed in the central nervous system (CNS. The main cause of MS is unknown. Nonetheless, the most probable theory is based on molecular mimicry, which suggests that some infections can activate T cells against brain auto-antigens like myelin proteolipid protein (PLP and initiate the disease cascade. This study is conducted to evaluate the activatory effects of PLP58-74 on T lymphocytes and humoral immunity. PLP58-74 was considered as an immunodominant epitope candidate of PLP using bioinformatics tools. Patients and healthy individuals’ peripheral blood mononuclear cells (PBMCs were treated with PLP58-74 and its proliferative effects were evaluated through assessing proliferating cell nuclear antigen (PCNA gene expression changes by real time PCR and immunocytochemistry assay. Finally, the rate of CD4+ and CD8+ T cells were assessed by flowcytometry. ELISA was also performed to measure anti PLP58-74 antibody in patients’ serum. PLP58-74 induced proliferation in patients’ PBMCs while it did not influence PBMCs of healthy individuals. CD4+ T cells were the main activated cells in reaction to PLP58-74 which increased from 22% to 39.91%. In addition, immune assay showed threefold increase in specific anti PLP58-74 IgG in patients compared to healthy controls. Results showed that PLP58-74 can stimulate CD4+ T cells and humoral immunity. Therefore it seems that the epitopes of some microorganisms mimicking PLP such as PLP58-74 might have a potential role in the initiation of MS. 

  17. INNOVATIVE STRATEGIES TO IDENTIFY M. TUBERCULOSIS ANTIGENS AND EPITOPES USING GENOME-WIDE ANALYSES

    Directory of Open Access Journals (Sweden)

    Annemieke eGeluk

    2014-06-01

    Full Text Available In view of the fact that only a small part of the Mtb expressome has been explored for identification of antigens capable of activating human T-cell responses, which is critically required for the design of better TB vaccination strategies, more emphasis should be placed on innovative ways to discover new Mtb antigens and explore their function at the several stages of infection. Better protective antigens for TB vaccines are urgently needed, also in view of the disappointing results of the MVA85 vaccine which failed to induce additional protection in BCG vaccinated infants [54]. Moreover, immune responses to relevant antigens may be useful to identify TB-specific biomarker signatures. Here we describe the potency of novel tools and strategies to reveal such Mtb antigens. Using proteins specific for different Mtb infection phases, many new antigens of the latency-associated Mtb DosR regulon as well as Rpf proteins, associated with resuscitating TB, were discovered that were recognized by CD4+ and CD8+ T-cells. Furthermore, by employing MHC binding algorithms and bioinformatics combined with high throughput human T-cell screens and tetramers, HLA-class Ia restricted poly-functional CD8+ T-cells were identified in TB patients. Comparable methods, led to the identification of HLA-E-restricted Mtb epitopes recognized by CD8+ T-cells. A genome-wide unbiased antigen discovery approach was applied to analyse the in vivo Mtb gene expression profiles in the lungs of mice, resulting in the identification of IVE-TB antigens, which are expressed during infection in the lung, the main target organ of Mtb. IVE-TB antigens induce strong T cell responses in long-term latently Mtb infected individuals, and represent an interesting new group of TB antigens for vaccination. In summary, new tools have helped expand our view on the Mtb antigenome involved in human cellular immunity and provided new candidates for TB vaccination.

  18. T-cell epitope vaccine design by immunoinformatics.

    Science.gov (United States)

    Patronov, Atanas; Doytchinova, Irini

    2013-01-08

    Vaccination is generally considered to be the most effective method of preventing infectious diseases. All vaccinations work by presenting a foreign antigen to the immune system in order to evoke an immune response. The active agent of a vaccine may be intact but inactivated ('attenuated') forms of the causative pathogens (bacteria or viruses), or purified components of the pathogen that have been found to be highly immunogenic. The increased understanding of antigen recognition at molecular level has resulted in the development of rationally designed peptide vaccines. The concept of peptide vaccines is based on identification and chemical synthesis of B-cell and T-cell epitopes which are immunodominant and can induce specific immune responses. The accelerating growth of bioinformatics techniques and applications along with the substantial amount of experimental data has given rise to a new field, called immunoinformatics. Immunoinformatics is a branch of bioinformatics dealing with in silico analysis and modelling of immunological data and problems. Different sequence- and structure-based immunoinformatics methods are reviewed in the paper.

  19. Novel CD8(+) cytotoxic T cell epitopes in bovine leukemia virus with cattle.

    Science.gov (United States)

    Bai, Lanlan; Takeshima, Shin-Nosuke; Isogai, Emiko; Kohara, Junko; Aida, Yoko

    2015-12-16

    Bovine leukemia virus (BLV) is associated with enzootic bovine leukosis and is closely related to human T cell leukemia virus (HTLV). The cytotoxic T lymphocyte (CTL) plays a key role in suppressing the progression of disease caused by BLV. T and B cell epitopes in BLV have been studied, but CD8(+) CTL epitopes remain poorly understood. We used a library of 115 synthetic peptides covering the entirety of the Env proteins (gp51 and gp30), the Gag proteins (p15, p24, and p12), and the Tax protein of BLV to identify 11 novel CD8(+) T cell epitopes (gp51N5, gp51N11, gp51N12, gp30N5, gp30N6, gp30N8, gp30N16, tax16, tax18, tax19, and tax20) in four calves experimentally infected with BLV. The number of CD8(+) T cell epitopes that could be identified in each calf correlated with the BLV proviral load. Interestingly, among the 11 epitopes identified, only gp51N11 was capable of inducing CD8(+) T cell-mediated cytotoxicity in all four calves, but it is not a suitable vaccine target because it shows a high degree of polymorphism according to the Wu-Kabat variability index. By contrast, no CTL epitopes were identified from the Gag structural protein. In addition, several epitopes were obtained from gp30 and Tax, indicating that cellular immunity against BLV is strongly targeted to these proteins. CD8(+) CTL epitopes from gp30 and Tax were less polymorphic than epitopes from. Indeed, peptides tax16, tax18, tax19, and tax20 include a leucine-rich activation domain that encompasses a transcriptional activation domain, and the gp30N16 peptide contains a proline-rich region that interacts with a protein tyrosine phosphatase SHP1 to regulate B cell activation. Moreover, at least one CD8(+) CTL epitope derived from gp30 was identified in each of the four calves. These results indicate that BLV gp30 may be the best candidate for the development of a BLV vaccine.

  20. Dengue virus antibody database: Systematically linking serotype-specificity with epitope mapping in dengue virus

    Science.gov (United States)

    Chaudhury, Sidhartha; Gromowski, Gregory D.; Ripoll, Daniel R.; Khavrutskii, Ilja V.; Desai, Valmik; Wallqvist, Anders

    2017-01-01

    Background A majority infections caused by dengue virus (DENV) are asymptomatic, but a higher incidence of severe illness, such as dengue hemorrhagic fever, is associated with secondary infections, suggesting that pre-existing immunity plays a central role in dengue pathogenesis. Primary infections are typically associated with a largely serotype-specific antibody response, while secondary infections show a shift to a broadly cross-reactive antibody response. Methods/Principal findings We hypothesized that the basis for the shift in serotype-specificity between primary and secondary infections can be found in a change in the antibody fine-specificity. To investigate the link between epitope- and serotype-specificity, we assembled the Dengue Virus Antibody Database, an online repository containing over 400 DENV-specific mAbs, each annotated with information on 1) its origin, including the immunogen, host immune history, and selection methods, 2) binding/neutralization data against all four DENV serotypes, and 3) epitope mapping at the domain or residue level to the DENV E protein. We combined epitope mapping and activity information to determine a residue-level index of epitope propensity and cross-reactivity and generated detailed composite epitope maps of primary and secondary antibody responses. We found differing patterns of epitope-specificity between primary and secondary infections, where secondary responses target a distinct subset of epitopes found in the primary response. We found that secondary infections were marked with an enhanced response to cross-reactive epitopes, such as the fusion-loop and E-dimer region, as well as increased cross-reactivity in what are typically more serotype-specific epitope regions, such as the domain I-II interface and domain III. Conclusions/Significance Our results support the theory that pre-existing cross-reactive memory B cells form the basis for the secondary antibody response, resulting in a broadening of the response

  1. Stable, conserved outer membrane epitope of strains of Haemophilus influenzae biogroup aegyptius associated with Brazilian purpuric fever.

    OpenAIRE

    Lesse, A J; Gheesling, L L; Bittner, W E; Myers, S.D.; Carlone, G M

    1992-01-01

    Brazilian purpuric fever is a rapidly fatal childhood disease associated with a clonal strain of Haemophilus influenzae biogroup aegyptius. We describe a conserved, surface-exposed epitope present on 95% of H. influenzae biogroup aegyptius isolates that are associated with Brazilian purpuric fever. This epitope, defined by reaction with the monoclonal antibody 8G3, is on or associated with the 48-kDa heat-modifiable P1 protein. The epitope is absent on strains of H. influenzae biogroup aegypt...

  2. TLR5-dependent immunogenicity of a recombinant fusion protein containing an immunodominant epitope of malarial circumsporozoite protein and the FliC flagellin of Salmonella Typhimurium

    Directory of Open Access Journals (Sweden)

    Ariane Guglielmi Ariza Camacho

    2011-08-01

    Full Text Available Recently, we described the improved immunogenicity of new malaria vaccine candidates based on the expression of fusion proteins containing immunodominant epitopes of merozoites and Salmonella enterica serovar Typhimurium flagellin (FliC protein as an innate immune agonist. Here, we tested whether a similar strategy, based on an immunodominant B-cell epitope from malaria sporozoites, could also generate immunogenic fusion polypeptides. A recombinant His6-tagged FliC protein containing the C-terminal repeat regions of the VK210 variant of Plasmodium vivax circumsporozoite (CS protein was constructed. This recombinant protein was successfully expressed in Escherichia coli as soluble protein and was purified by affinity to Ni-agarose beads followed by ion exchange chromatography. A monoclonal antibody specific for the CS protein of P. vivax sporozoites (VK210 was able to recognise the purified protein. C57BL/6 mice subcutaneously immunised with the recombinant fusion protein in the absence of any conventional adjuvant developed protein-specific systemic antibody responses. However, in mice genetically deficient in expression of TLR5, this immune response was extremely low. These results extend our previous observations concerning the immunogenicity of these recombinant fusion proteins and provide evidence that the main mechanism responsible for this immune activation involves interactions with TLR5, which has not previously been demonstrated for any recombinant FliC fusion protein.

  3. Quinine-dependent antibodies bind a restricted set of epitopes on the glycoprotein Ib-IX complex: Characterization of the epitopes

    NARCIS (Netherlands)

    Burgess, Janette K.; Lopez, Jose A.; Berndt, Michael C.; Dawes, Ian; Chesterman, Colin N.; Chong, Beng H.

    1998-01-01

    Severe immune thrombocytopenia is an idiosyncratic complication of quinine therapy. Although in most cases the responsible antibody is directed against platelet membrane glycoprotein (GP) Ib-IX, specificity for GPIIb- IIIa or both epitopes has also been reported. The objective of this study was to c

  4. Quinine-dependent antibodies bind a restricted set of epitopes on the glycoprotein Ib-IX complex : characterization of the epitopes

    NARCIS (Netherlands)

    Burgess, J K; Lopez, J A; Berndt, M C; Dawes, I; Chesterman, C N; Chong, B H

    1998-01-01

    Severe immune thrombocytopenia is an idiosyncratic complication of quinine therapy. Although in most cases the responsible antibody is directed against platelet membrane glycoprotein (GP) Ib-IX, specificity for GPIIb-IIIa or both epitopes has also been reported. The objective of this study was to ch

  5. The Role of Glutamic or Aspartic Acid in Position Four of the Epitope Binding Motif and Thyrotropin Receptor-Extracellular Domain Epitope Selection in Graves' Disease

    Science.gov (United States)

    Inaba, Hidefumi; Martin, William; Ardito, Matt; De Groot, Anne Searls; De Groot, Leslie J.

    2010-01-01

    Context: Development of Graves' disease (GD) is related to HLA-DRB1*0301 (DR3),and more specifically to arginine at position 74 of the DRB1 molecule. The extracellular domain (ECD) of human TSH receptor (hTSH-R) contains the target antigen. Objective and Design: We analyzed the relation between hTSH-R-ECD peptides and DR molecules to determine whether aspartic acid (D) or glutamic acid (E) at position four in the binding motif influenced selection of functional epitopes. Results: Peptide epitopes from TSH-R-ECD with D or E in position four (D/E+) had higher affinity for binding to DR3 than peptides without D/E (D/E−) (IC50 29.3 vs. 61.4, P = 0.0024). HLA-DR7, negatively correlated with GD, and DRB1*0302 (HLA-DR18), not associated with GD, had different profiles of epitope binding. Toxic GD patients who are DR3+ had higher responses to D/E+ peptides than D/E− peptides (stimulation index 1.42 vs. 1.22, P = 0.028). All DR3+ GD patients (toxic + euthyroid) had higher responses, with borderline significance (Sl; 1.32 vs. 1.18, P = 0.051). Splenocytes of DR3 transgenic mice immunized to TSH-R-ECD responded to D/E+ peptides more than D/E− peptides (stimulation index 1.95 vs. 1.69, P = 0.036). Seven of nine hTSH-R-ECD peptide epitopes reported to be reactive with GD patients' peripheral blood mononuclear cells contain binding motifs with D/E at position four. Conclusions: TSH-R-ECD epitopes with D/E in position four of the binding motif bind more strongly to DRB1*0301 than epitopes that are D/E− and are more stimulatory to GD patients' peripheral blood mononuclear cells and to splenocytes from mice immunized to hTSH-R. These epitopes appear important in immunogenicity to TSH-R due to their favored binding to HLA-DR3, thus increasing presentation to T cells. PMID:20392871

  6. The first human epitope map of the alphaviral E1 and E2 proteins reveals a new E2 epitope with significant virus neutralizing activity.

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    Ann R Hunt

    Full Text Available BACKGROUND: Venezuelan equine encephalitis virus (VEEV is responsible for VEE epidemics that occur in South and Central America and the U.S. The VEEV envelope contains two glycoproteins E1 (mediates cell membrane fusion and E2 (binds receptor and elicits virus neutralizing antibodies. Previously we constructed E1 and E2 epitope maps using murine monoclonal antibodies (mMAbs. Six E2 epitopes (E2(c,d,e,f,g,h bound VEEV-neutralizing antibody and mapped to amino acids (aa 182-207. Nothing is known about the human antibody repertoire to VEEV or epitopes that engage human virus-neutralizing antibodies. There is no specific treatment for VEE; however virus-neutralizing mMAbs are potent protective and therapeutic agents for mice challenged with VEEV by either peripheral or aerosol routes. Therefore, fully human MAbs (hMAbs with virus-neutralizing activity should be useful for prevention or clinical treatment of human VEE. METHODS: We used phage-display to isolate VEEV-specific hFabs from human bone marrow donors. These hFabs were characterized by sequencing, specificity testing, VEEV subtype cross-reactivity using indirect ELISA, and in vitro virus neutralization capacity. One E2-specific neutralizing hFAb, F5n, was converted into IgG, and its binding site was identified using competitive ELISA with mMAbs and by preparing and sequencing antibody neutralization-escape variants. FINDINGS: Using 11 VEEV-reactive hFabs we constructed the first human epitope map for the alphaviral surface proteins E1 and E2. We identified an important neutralization-associated epitope unique to the human immune response, E2 aa115-119. Using a 9 A resolution cryo-electron microscopy map of the Sindbis virus E2 protein, we showed the probable surface location of this human VEEV epitope. CONCLUSIONS: The VEEV-neutralizing capacity of the hMAb F5 nIgG is similar to that exhibited by the humanized mMAb Hy4 IgG. The Hy4 IgG has been shown to limit VEEV infection in mice both

  7. Beyond HLA-A*0201: new HLA-transgenic nonobese diabetic mouse models of type 1 diabetes identify the insulin C-peptide as a rich source of CD8+ T cell epitopes.

    Science.gov (United States)

    Antal, Zoltan; Baker, Jason C; Smith, Carla; Jarchum, Irene; Babad, Jeffrey; Mukherjee, Gayatri; Yang, Yang; Sidney, John; Sette, Alessandro; Santamaria, Pere; DiLorenzo, Teresa P

    2012-06-01

    Type 1 diabetes is an autoimmune disease characterized by T cell responses to β cell Ags, including insulin. Investigations employing the NOD mouse model of the disease have revealed an essential role for β cell-specific CD8(+) T cells in the pathogenic process. As CD8(+) T cells specific for β cell Ags are also present in patients, these reactivities have the potential to serve as therapeutic targets or markers for autoimmune activity. NOD mice transgenic for human class I MHC molecules have previously been employed to identify T cell epitopes having important relevance to the human disease. However, most studies have focused exclusively on HLA-A*0201. To broaden the reach of epitope-based monitoring and therapeutic strategies, we have looked beyond this allele and developed NOD mice expressing human β(2)-microglobulin and HLA-A*1101 or HLA-B*0702, which are representative members of the A3 and B7 HLA supertypes, respectively. We have used islet-infiltrating T cells spontaneously arising in these strains to identify β cell peptides recognized in the context of the transgenic HLA molecules. This work has identified the insulin C-peptide as an abundant source of CD8(+) T cell epitopes. Responses to these epitopes should be of considerable utility for immune monitoring, as they cannot reflect an immune reaction to exogenously administered insulin, which lacks the C-peptide. Because the peptides bound by one supertype member were found to bind certain other members also, the epitopes identified in this study have the potential to result in therapeutic and monitoring tools applicable to large numbers of patients and at-risk individuals.

  8. Construction of recombinant Mip-FlaA dominant epitope vaccine against Legionella pneumophila and evaluation of the immunogenicity and protective immunity.

    Science.gov (United States)

    He, Jinlei; Zhang, Junrong; He, Yanxia; Huang, Fan; Li, Jiao; Chen, Qiwei; Chen, Dali; Chen, Jianping

    2016-02-01

    Legionnaires' disease, a kind of systemic disease with pneumonia as the main manifestation, is caused by Legionella pneumophila (L. pneumophila). In order to prevent the disease, we optimized Mip and FlaA, the virulence factors of L. pneumophila, to design recombinant Mip-FlaA dominant epitope vaccine against the pathogen. Firstly, the coding sequences of mip and flaA were optimized by DNAStar software and Expasy protein analysis system, and then, the tertiary structure and function of recombinant Mip-FlaA were predicted by PHYRE2 Protein Fold Recognition Server. After that, the optimized mip, flaA and mip-flaA were cloned, expressed and purified, and the proteins were used as dominant epitope vaccines to immunize BABL/c mice. Moreover, the IgG titers, histological changes in lung and the level of TNF-α, IFN-γ, IL-6 and IL-1β were detected to reflect the immunogenicity and protective immunity of the vaccines. The results of SDS-PAGE and Western blot proved the recombinant Mip-FlaA was successfully expressed. ELISA results of IgG titers and these cytokines showed Mip-FlaA group was capable to induce the strongest immune response, compared to PBS, Mip and FlaA groups. In addition, histopathology analysis demonstrated the mice immunized with Mip-FlaA showed better immune protection. Therefore, the work indicated that the above-described biological tools were useful in optimization of epitope vaccine. Antigenic characterization and immune protection of recombinant Mip-FlaA would be of great value in understanding the immunopathogenesis of the disease and in developing possible vaccine against the pathogen.

  9. Receptor epitope usage by an interleukin-5 mimetic peptide.

    Science.gov (United States)

    Ishino, Tetsuya; Urbina, Cecilia; Bhattacharya, Madhushree; Panarello, Dominick; Chaiken, Irwin

    2005-06-17

    The cyclic peptide AF17121 is a library-derived antagonist for human interleukin-5 (IL5) receptor alpha (IL5Ralpha) and inhibits IL5 activity. Our previous results have demonstrated that the sixth arginine residue of the peptide is crucial for the inhibitory effect and that several acidic residues in the N- and C-terminal regions also make a contribution, although to a lesser extent (Ruchala, P., Varadi, G., Ishino, T., Scibek, J., Bhattacharya, M., Urbina, C., Van Ryk, D., Uings, I., and Chaiken, I. (2004) Biopolymers 73, 556-568). However, the recognition mechanism of the receptor has remained unresolved. In this study, AF17121 was fused to thioredoxin by recombinant DNA techniques and examined for IL5Ralpha interaction using a surface plasmon resonance biosensor method. Kinetic analysis revealed that the dissociation rate of the peptide.receptor complex is comparable with that of the cytokine.receptor complex. The fusion peptide competed with IL5 for both biological function and interaction with IL5Ralpha, indicating that the binding sites on the receptor are shared by AF17121 and IL5. To define the epitope residues for AF17121, we defined its binding footprint on IL5Ralpha by alanine substitution of Asp(55), Asp(56), Glu(58), Lys(186), Arg(188), and Arg(297) of the receptor. Marked effects on the interaction were observed in all three fibronectin type III domains of IL5Ralpha, in particular Asp(55), Arg(188), and Arg(297) in the D1, D2, and D3 domains, respectively. This footprint represents a significant subset of that for IL5 binding. The fact that AF17121 mimics the receptor binding capability of IL5 but antagonizes biological function evokes several models for how IL5 induces activation of the multisubunit receptor system.

  10. Epitope Mapping of Metuximab on CD147 Using Phage Display and Molecular Docking

    Directory of Open Access Journals (Sweden)

    Bifang He

    2013-01-01

    Full Text Available Metuximab is the generic name of Licartin, a new drug for radioimmunotherapy of hepatocellular carcinoma. Although it is known to be a mouse monoclonal antibody against CD147, the complete epitope mediating the binding of metuximab to CD147 remains unknown. We panned the Ph.D.-12 phage display peptide library against metuximab and got six mimotopes. The following bioinformatics analysis based on mimotopes suggested that metuximab recognizes a conformational epitope composed of more than 20 residues. The residues of its epitope may include T28, V30, K36, L38, K57, F74, D77, S78, D79, D80, Q81, G83, S86, N98, Q100, L101, H102, G103, P104, V131, P132, and K191. The homology modeling of metuximab and the docking of CD147 to metuximab were also performed. Based on the top one docking model, the epitope was predicted to contain 28 residues: AGTVFTTV (23–30, I37, D45, E84, V88, EPMGTANIQLH (92–102, VPP (131–133, Q164, and K191. Almost half of the residues predicted on the basis of mimotope analysis also appear in the docking result, indicating that both results are reliable. As the predicted epitopes of metuximab largely overlap with interfaces of CD147-CD147 interactions, a structural mechanism of metuximab is proposed as blocking the formation of CD147 dimer.

  11. Folding of matrix metalloproteinase-2 prevents endogenous generation of MHC class-I restricted epitope.

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    Virginie Renaud

    Full Text Available BACKGROUND: We previously demonstrated that the matrix metalloproteinase-2 (MMP-2 contained an antigenic peptide recognized by a CD8 T cell clone in the HLA-A*0201 context. The presentation of this peptide on class I molecules by human melanoma cells required a cross-presentation mechanism. Surprisingly, the classical endogenous processing pathway did not process this MMP-2 epitope. METHODOLOGY/PRINCIPAL FINDINGS: By PCR directed mutagenesis we showed that disruption of a single disulfide bond induced MMP-2 epitope presentation. By Pulse-Chase experiment, we demonstrated that disulfide bonds stabilized MMP-2 and impeded its degradation. Finally, using drugs, we documented that mutated MMP-2 epitope presentation used the proteasome and retrotranslocation complex. CONCLUSIONS/SIGNIFICANCE: These data appear crucial to us since they established the existence of a new inhibitory mechanism for the generation of a T cell epitope. In spite of MMP-2 classified as a self-antigen, the fact that cross-presentation is the only way to present this MMP-2 epitope underlines the importance to target this type of antigen in immunotherapy protocols.

  12. Epitope prediction based on random peptide library screening: benchmark dataset and prediction tools evaluation.

    Science.gov (United States)

    Sun, Pingping; Chen, Wenhan; Huang, Yanxin; Wang, Hongyan; Ma, Zhiqiang; Lv, Yinghua

    2011-06-16

    Epitope prediction based on random peptide library screening has become a focus as a promising method in immunoinformatics research. Some novel software and web-based servers have been proposed in recent years and have succeeded in given test cases. However, since the number of available mimotopes with the relevant structure of template-target complex is limited, a systematic evaluation of these methods is still absent. In this study, a new benchmark dataset was defined. Using this benchmark dataset and a representative dataset, five examples of the most popular epitope prediction software products which are based on random peptide library screening have been evaluated. Using the benchmark dataset, in no method did performance exceed a 0.42 precision and 0.37 sensitivity, and the MCC scores suggest that the epitope prediction results of these software programs are greater than random prediction about 0.09-0.13; while using the representative dataset, most of the values of these performance measures are slightly improved, but the overall performance is still not satisfactory. Many test cases in the benchmark dataset cannot be applied to these pieces of software due to software limitations. Moreover chances are that these software products are overfitted to the small dataset and will fail in other cases. Therefore finding the correlation between mimotopes and genuine epitope residues is still far from resolved and much larger dataset for mimotope-based epitope prediction is desirable.

  13. Epitope Prediction Based on Random Peptide Library Screening: Benchmark Dataset and Prediction Tools Evaluation

    Directory of Open Access Journals (Sweden)

    Yanxin Huang

    2011-06-01

    Full Text Available Epitope prediction based on random peptide library screening has become a focus as a promising method in immunoinformatics research. Some novel software and web-based servers have been proposed in recent years and have succeeded in given test cases. However, since the number of available mimotopes with the relevant structure of template-target complex is limited, a systematic evaluation of these methods is still absent. In this study, a new benchmark dataset was defined. Using this benchmark dataset and a representative dataset, five examples of the most popular epitope prediction software products which are based on random peptide library screening have been evaluated. Using the benchmark dataset, in no method did performance exceed a 0.42 precision and 0.37 sensitivity, and the MCC scores suggest that the epitope prediction results of these software programs are greater than random prediction about 0.09–0.13; while using the representative dataset, most of the values of these performance measures are slightly improved, but the overall performance is still not satisfactory. Many test cases in the benchmark dataset cannot be applied to these pieces of software due to software limitations. Moreover chances are that these software products are overfitted to the small dataset and will fail in other cases. Therefore finding the correlation between mimotopes and genuine epitope residues is still far from resolved and much larger dataset for mimotope-based epitope prediction is desirable.

  14. Sequence evolution of putative cytotoxic T cell epitopes in NS3 region of hepatitis C virus

    Institute of Scientific and Technical Information of China (English)

    Hua-Zhang Guo; Ying Yin; Wen-Liang Wang; Chuan-Shan Zhang; Tao Wang; Zhe Wang; Jing Zhang; Hong Cheng; Hai-Tao Wang

    2004-01-01

    AIM: Quasispecies of hepatitis C virus (HCV) are the foundation for rapid sequence evolution of HCV to evade immune surveillance of hosts. The consensus sequence evolution of a segment of HCV NS3 region, which encompasses putative cytotoxic T cell epitopes, was evaluated. METHODS: Three male patients, infected with HCV through multiple transfusions, were identified from clinical symptoms and monitored by aminotransferase for 60 months. Blood samples taken at months 0, 32, and 60 were used for viral RNA extraction. A segment of HCV NS3 region was amplified from the RNA extraction by RT-PCR and subjected to subcloning and sequencing. HLA types of these three patients were determined using complement-dependent microlymphocytotoxic assay. CTL epitopes were predicted using MHC binding motifs.RESULTS: No patient had clinical symptoms or elevation of aspartate/alanine aminotransferase. Two patients showed positive HCV PCR results at all 3 time points. The other one showed a positive HCV PCR result only at month O. A reported HLA-A2-restricted CTL epitope had no alteration in the HLA-A2-negative carrier over 60 months. In the HLA-A2-positive individuals, all the sequences from O month Oshowed an amber mutation on the initial codon of the epitope. Most changes of consensus sequences in the samepatient occurred on predicted cytotoxic T cell epitopes. CONCLUSION: Amber mutation and changes of consensussequence in HCV NS3 region may be related to viral immune escape.

  15. Characterization of Peptide Antibodies by Epitope Mapping Using Resin-Bound and Soluble Peptides.

    Science.gov (United States)

    Trier, Nicole Hartwig

    2015-01-01

    Characterization of peptide antibodies through identification of their target epitopes is of utmost importance. Understanding antibody specificity at the amino acid level provides the key to understand the specific interaction between antibodies and their epitopes and their use as research and diagnostic tools as well as therapeutic agents. This chapter describes a straightforward strategy for mapping of continuous peptide antibody epitopes using resin-bound and soluble peptides. The approach combines three different types of peptide sets for full characterization of peptide antibodies: (1) overlapping peptides, used to locate antigenic regions; (2) truncated peptides, used to identify the minimal peptide length required for antibody binding; and (3) substituted peptides, used to identify the key residues important for antibody binding and to determine the specific contribution of key residues. For initial screening resin-bound peptides are used for epitope estimation, while soluble peptides subsequently are used for fine mapping. The combination of resin-bound peptides and soluble peptides for epitope mapping provides a time-sparing and straightforward approach for characterization of peptide antibodies.

  16. Epitope mapping of metuximab on CD147 using phage display and molecular docking.

    Science.gov (United States)

    He, Bifang; Mao, Canquan; Ru, Beibei; Han, Hesong; Zhou, Peng; Huang, Jian

    2013-01-01

    Metuximab is the generic name of Licartin, a new drug for radioimmunotherapy of hepatocellular carcinoma. Although it is known to be a mouse monoclonal antibody against CD147, the complete epitope mediating the binding of metuximab to CD147 remains unknown. We panned the Ph.D.-12 phage display peptide library against metuximab and got six mimotopes. The following bioinformatics analysis based on mimotopes suggested that metuximab recognizes a conformational epitope composed of more than 20 residues. The residues of its epitope may include T28, V30, K36, L38, K57, F74, D77, S78, D79, D80, Q81, G83, S86, N98, Q100, L101, H102, G103, P104, V131, P132, and K191. The homology modeling of metuximab and the docking of CD147 to metuximab were also performed. Based on the top one docking model, the epitope was predicted to contain 28 residues: AGTVFTTV (23-30), I37, D45, E84, V88, EPMGTANIQLH (92-102), VPP (131-133), Q164, and K191. Almost half of the residues predicted on the basis of mimotope analysis also appear in the docking result, indicating that both results are reliable. As the predicted epitopes of metuximab largely overlap with interfaces of CD147-CD147 interactions, a structural mechanism of metuximab is proposed as blocking the formation of CD147 dimer.

  17. Construction and characterization of a recombinant fowlpox virus containing HIV-1 multi-epitope-p24 chimeric gene in mice

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    The epidemic of HIV/AIDS is sweeping across the world. It is of great importance to figure out new ways to curb this disease. Epitope-based vaccine is one of these solutions. In this study, a chimeric gene was obtained by combination of a designed HIV-1 multi-epitope gene (MEG) and HIV-1 p24 gene. A re- combinant plasmid pUTA2-MEGp24 was then constructed by inserting MEGp24 gene into the down- stream of the promoter (ATI-P7.5×20) of fowlpox virus (FPV) transfer vector pUTA2. The recombinant plasmid and wild-type FPV 282E4 strain were then co-transfected into CEF cells and homologous re- combination occurred. A recombinant virus expressing HIV-1 protein MEGp24 was screened by ge- nome PCR and Western blot assay. Large scale preparation and purification of the recombinant fowl- pox virus (rFPV) were then carried out. BALB/c mice were immunized intramuscularly with the rFPV for three times on day 0, 14 and 42. Mice were executed and sampled one week after the third inoculation. Anti-HIV-1 antibody in serum and Th1 cytokines in the supernatant of cultured spleen cells were as- sayed by ELISA. The count of T lymphocyte subsets and the CTL activity of spleen lymphocytes were analyzed by flow cytometry and lactate dehydrogenase (LDH) release assay, respectively. The results showed that HIV-1 specific antibody in serum and increased T lymphocyte subsets (CD4+ T, CD8+ T) were detected in the immunization group. CTL target-killing activity and higher secretion of Th1 cyto- kines (IFN-γ and IL-2) of spleen lymphocytes stimulated by H-2d-restricted CTL peptide were observed in immunized mice. We concluded that the rFPV may induce HIV-1 specific immunity especially cellular immunity in mice.

  18. Construction and characterization of a recombinant fowlpox virus containing HIV-1 multi-epitope-p24 chimeric gene in mice

    Institute of Scientific and Technical Information of China (English)

    ZHANG LiShu; JIN NingYi; SONG YingJin; WANG Hong; MA HeWen; LI ZiJian; JIANG WenZheng

    2007-01-01

    The epidemic of HIV/AIDS is sweeping across the world. It is of great importance to figure out new ways to curb this disease. Epitope-based vaccine is one of these solutions. In this study, a chimeric gene was obtained by combination of a designed HIV-1 multi-epitope gene (MEG) and HIV-1 p24 gene. A recombinant plasmid pUTA2-MEGp24 was then constructed by inserting MEGp24 gene into the down-stream of the promoter (ATI-P7.5×20) of fowlpox virus (FPV) transfer vector pUTA2. The recombinant plasmid and wild-type FPV 282E4 strain were then co-transfected into CEF cells and homologous recombination occurred. A recombinant virus expressing HIV-1 protein MEGp24 was screened by genome PCR and Western blot assay. Large scale preparation and purification of the recombinant fowlpox virus (rFPV) were then carried out. BALB/c mice were immunized intramuscularly with the rFPV for three times on day 0, 14 and 42. Mice were executed and sampled one week after the third inoculation.Anti-HIV-1 antibody in serum and Th1 cytokines in the supernatant of cultured spleen cells were assayed by ELISA. The count of T lymphocyte subsets and the CTL activity of spleen lymphocytes were analyzed by flow cytometry and lactate dehydrogenase (LDH) release assay, respectively. The results showed that HIV-1 specific antibody in serum and increased T lymphocyte subsets (CD4+ T, CD8+ T)were detected in the immunization group. CTL target-killing activity and higher secretion of Th1 cytokines (IFN-Y and IL-2) of spleen lymphocytes stimulated by H-2d-restricted CTL peptide were observed in immunized mice.We concluded that the rFPV may induce HIV-1 specific immunity especially cellular immunity in mice.

  19. A human multi-epitope recombinant vaccinia virus as a universal T cell vaccine candidate against influenza virus.

    Directory of Open Access Journals (Sweden)

    Alan G Goodman

    Full Text Available There is a need to develop a universal vaccine against influenza virus infection to avoid developing new formulations of a seasonal vaccine each year. Many of the vaccine strategies for a universal vaccine target strain-conserved influenza virus proteins, such as the matrix, polymerase, and nucleoproteins, rather than the surface hemagglutinin and neuraminidase proteins. In addition, non-disease-causing viral vectors are a popular choice as a delivery system for the influenza virus antigens. As a proof-of-concept, we have designed a novel influenza virus immunogen based on the NP backbone containing human T cell epitopes for M1, NS1, NP, PB1 and PA proteins (referred as NPmix as well as a construct containing the conserved regions of influenza virus neuraminidase (N-terminal and hemagglutinin (C-terminal (referred as NA-HA. DNA vectors and vaccinia virus recombinants expressing NPmix (WR-NP or both NPmix plus NA-HA (WR-flu in the cytosol were tested in a heterologous DNA-prime/vaccinia virus-boost vaccine regimen in mice. We observed an increase in the number of influenza virus-specific IFNγ-secreting splenocytes, composed of populations marked by CD4(+ and CD8(+ T cells producing IFNγ or TNFα. Upon challenge with influenza virus, the vaccinated mice exhibited decreased viral load in the lungs and a delay in mortality. These findings suggest that DNA prime/poxvirus boost with human multi-epitope recombinant influenza virus proteins is a valid approach for a general T-cell vaccine to protect against influenza virus infection.

  20. Antibodies Directed against a Peptide Epitope of a Klebsiella pneumoniae-Derived Protein Are Present in Ankylosing Spondylitis

    Science.gov (United States)

    Tinazzi, Elisa; Moretta, Francesca; D’Angelo, Salvatore; Olivieri, Ignazio; Lunardi, Claudio

    2017-01-01

    Ankylosing spondylitis (AS) is a chronic inflammatory arthritis of unknown origin. Its autoimmune origin has been suggested but never proven. Several reports have implicated Klebsiella pneumoniae as a triggering or perpetuating factor in AS; however, its role in the disease pathogenesis remains debated. Moreover, despite extensive investigations, a biomarker for AS has not yet been identified. To clarify these issues, we screened a random peptide library with pooled IgGs obtained from 40 patients with AS. A peptide (AS peptide) selected from the library was recognized by serum IgGs from 170 of 200 (85%) patients with AS but not by serum specimens from 100 healthy controls. Interestingly, the AS peptide shows a sequence similarity with several molecules expressed at the fibrocartilaginous sites that are primarily involved in the AS inflammatory process. Moreover, the peptide is highly homologous to a Klebsiella pneumoniae dipeptidase (DPP) protein. The antibody affinity purified against the AS peptide recognizes the autoantigens and the DPP protein. Furthermore, serum IgG antibodies against the Klebsiella DPP121-145 peptide epitope were detected in 190 of 200 patients with AS (95%), 3 of 200 patients with rheumatoid arthritis (1.5%) and only 1 of 100 (1%) patients with psoriatic arthritis. Such reactivity was not detected in healthy control donors. Our results show that antibodies directed against an epitope of a Klebsiella pneumoniae-derived protein are present in nearly all patients with AS. In the absence of serological biomarkers for AS, such antibodies may represent a useful tool in the diagnosis of the disease. PMID:28135336

  1. ArrayPitope: Automated Analysis of Amino Acid Substitutions for Peptide Microarray-Based Antibody Epitope Mapping

    Science.gov (United States)

    Hansen, Christian Skjødt; Østerbye, Thomas; Marcatili, Paolo; Lund, Ole; Buus, Søren

    2017-01-01

    Identification of epitopes targeted by antibodies (B cell epitopes) is of critical importance for the development of many diagnostic and therapeutic tools. For clinical usage, such epitopes must be extensively characterized in order to validate specificity and to document potential cross-reactivity. B cell epitopes are typically classified as either linear epitopes, i.e. short consecutive segments from the protein sequence or conformational epitopes adapted through native protein folding. Recent advances in high-density peptide microarrays enable high-throughput, high-resolution identification and characterization of linear B cell epitopes. Using exhaustive amino acid substitution analysis of peptides originating from target antigens, these microarrays can be used to address the specificity of polyclonal antibodies raised against such antigens containing hundreds of epitopes. However, the interpretation of the data provided in such large-scale screenings is far from trivial and in most cases it requires advanced computational and statistical skills. Here, we present an online application for automated identification of linear B cell epitopes, allowing the non-expert user to analyse peptide microarray data. The application takes as input quantitative peptide data of fully or partially substituted overlapping peptides from a given antigen sequence and identifies epitope residues (residues that are significantly affected by substitutions) and visualize the selectivity towards each residue by sequence logo plots. Demonstrating utility, the application was used to identify and address the antibody specificity of 18 linear epitope regions in Human Serum Albumin (HSA), using peptide microarray data consisting of fully substituted peptides spanning the entire sequence of HSA and incubated with polyclonal rabbit anti-HSA (and mouse anti-rabbit-Cy3). The application is made available at: www.cbs.dtu.dk/services/ArrayPitope. PMID:28095436

  2. Aspartate-β-hydroxylase Induces Epitope-specific T Cell Responses in Hepatocellular Carcinoma

    Science.gov (United States)

    Tomimaru, Yoshito; Mishra, Sasmita; Safran, Howard; Charpentier, Kevin P.; Martin, William; De Groot, Anne S.; Gregory, Stephen H.; Wands, Jack R.

    2015-01-01

    Hepatocellular carcinoma (HCC) has a poor prognosis due to high recurrence rate. Aspartate-β-hydroxylase (ASPH) is a highly conserved transmembrane protein, which is over expressed in HCC and promotes a malignant phenotype. The capability of ASPH protein-derived HLA Class I and II peptides to generate antigen specific CD4+ and CD8+ immune responses is unknown. Therefore, these studies aim to define the epitope specific components required for a peptide based candidate vaccine. Monocyte-derived dendritic cells (DCs) generated from the peripheral blood mononuclear cells (PBMCs) of HCC patients were loaded with ASPH protein. Helper CD4+ T cells and CD8+ cytotoxic T lymphocytes (CTLs) were co-incubated with the DCs; T cell activation was evaluated by flow cytometric analysis. Immunoinformatics tools were used to predict HLA class I- and class II-restricted ASPH sequences, and the corresponding peptides were synthesized. The immunogenicity of each peptide in cultures of human PBMCs was determined by IFN-γ ELISpot assay. ASPH protein-loaded DCs activated both CD4+ and CD8+ T cells contained within the PBMC population derived from HCC patients. Furthermore, the predicted HLA class I- and class II-restricted ASPH peptides were significantly immunogenic. Both HLA class I- and class II-restricted peptides derived from ASPH induce T cell activation in HCC. We observed that ASPH protein and related peptides were highly immunogenic in patients with HCC and produce the type of cellular immune responses required for generation of anti-tumor activity. PMID:25629522

  3. CD133 protein N-glycosylation processing contributes to cell surface recognition of the primitive cell marker AC133 epitope.

    Science.gov (United States)

    Mak, Anthony B; Blakely, Kim M; Williams, Rashida A; Penttilä, Pier-Andrée; Shukalyuk, Andrey I; Osman, Khan T; Kasimer, Dahlia; Ketela, Troy; Moffat, Jason

    2011-11-25

    The AC133 epitope expressed on the CD133 glycoprotein has been widely used as a cell surface marker of numerous stem cell and cancer stem cell types. It has been recently proposed that posttranslational modification and regulation of CD133 may govern cell surface AC133 recognition. Therefore, we performed a large scale pooled RNA interference (RNAi) screen to identify genes involved in cell surface AC133 expression. Gene hits could be validated at a rate of 70.5% in a secondary assay using an orthogonal RNAi system, demonstrating that our primary RNAi screen served as a powerful genetic screening approach. Within the list of hits from the primary screen, genes involved in N-glycan biosynthesis were significantly enriched as determined by Ingenuity Canonical Pathway analyses. Indeed, inhibiting biosynthesis of the N-glycan precursor using the small molecule tunicamycin or inhibiting its transfer to CD133 by generating N-glycan-deficient CD133 mutants resulted in undetectable cell surface AC133. Among the screen hits involved in N-glycosylation were genes involved in complex N-glycan processing, including the poorly characterized MGAT4C, which we demonstrate to be a positive regulator of cell surface AC133 expression. Our study identifies a set of genes involved in CD133 N-glycosylation as a direct contributing factor to cell surface AC133 recognition and provides biochemical evidence for the function and structure of CD133 N-glycans.

  4. Allergenic study and epitope analysis of Indo-American hybrid Tomatoes

    Directory of Open Access Journals (Sweden)

    Majeed Jamakhani

    2016-09-01

    Full Text Available Food allergies have high negative impact on nutritional balance of human body. Allergic response is seen high to vegetables such as tomatoes, capsicum and spinach, next to fish, eggs and nuts. Allergen molecules and epitope regions of Indo- American hybrid variety tomatoes is not well studied. The present work shows the presence of putative allergen molecules in two Indio-American hybrid tomatoes, which are identified in 7 patient sera by SDS - Immunoblotting method and further identification of epitopes, putative peptides and cross reactivity of these putative allergens by Immunoinformatics tools. Linear B-cell epitopes predicted by 3 combined immunoinformatics tools viz. DNASTAR protean, ABC pred and IEDB Bipred. Cross reactivity of these allergens determined by sequence alignment method against the allergen online database and Allermatch databases, which shows significant cross reactivity to Capsicum annum, Hevea brasiliensi, archis hypogaea, pollen, actinidia chinesis and prunus avium.

  5. Identification of immunogenic HLA-B7 "Achilles' heel" epitopes within highly conserved regions of HIV

    DEFF Research Database (Denmark)

    De Groot, Anne S; Rivera, Daniel S; McMurry, Julie A;

    2008-01-01

    previously described as restricted by B7. The HLA-B7 restricted epitopes discovered using this in silico screening approach are highly conserved across strains and clades of HIV as well as conserved in the HIV genome over the 20 years since HIV-1 isolates were first sequenced. This study demonstrates......Genetic polymorphisms in class I human leukocyte antigen molecules (HLA) have been shown to determine susceptibility to HIV infection as well as the rate of progression to AIDS. In particular, the HLA-B7 supertype has been shown to be associated with high viral loads and rapid progression...... to disease. Using a multiplatform in silico/in vitro approach, we have prospectively identified 45 highly conserved, putative HLA-B7 restricted HIV CTL epitopes and evaluated them in HLA binding and ELISpot assays. All 45 epitopes (100%) bound to HLA-B7 in cell-based HLA binding assays: 28 (62%) bound...

  6. High efficiency recovery and epitope specific sorting of an scFv yeast display library

    Energy Technology Data Exchange (ETDEWEB)

    Siegel, Robert W.; Coleman, James R.; Miller, Keith D.; Feldhaus, Michael

    2004-03-01

    In order to more productively utilize the rich source of antigen specific reagents present in the previously described non-immune scFv yeast display library (Feldhaus et al., 2003) one must be able to efficiently isolate and characterize clones within the library. To this end, we have developed and validated a magnetic bead sorting technique utilizing the Miltenyi MacsTm system to recover greater than 90% of the antigen specific clones present in the library. In combination with flow cytometry, we rapidly reduced diversity and enriched for antigen specific clones in three rounds of selection. Furthermore, we demonstrate the use of pre-existing monoclonal antibodies (mAbs) for antigen labeling and subsequent flow cytometric sorting and characterization of epitope specific scFv. Combining these two improvements in library screening allowed isolation and characterization of 3 epitope specific scFv (including a previously uncharacterized epitope) to a 6 kd protein, epidermal growth factor EGF.

  7. Microwave-based treatments of wheat kernels do not abolish gluten epitopes implicated in celiac disease.

    Science.gov (United States)

    Gianfrani, Carmen; Mamone, Gianfranco; la Gatta, Barbara; Camarca, Alessandra; Di Stasio, Luigia; Maurano, Francesco; Picascia, Stefania; Capozzi, Vito; Perna, Giuseppe; Picariello, Gianluca; Di Luccia, Aldo

    2017-03-01

    Microwave based treatment (MWT) of wet wheat kernels induced a striking reduction of gluten, up to gluten-free. In contrast, analysis of gluten peptides by G12 antibody-based ELISA, mass spectrometry-based proteomics and in vitro assay with T cells of celiac subjects, indicated no difference of antigenicity before and after MWT. SDS-PAGE analysis and Raman spectroscopy demonstrated that MWT simply induced conformational modifications, reducing alcohol solubility of gliadins and altering the access of R5-antibody to the gluten epitopes. Thus, MWT neither destroys gluten nor modifies chemically the toxic epitopes, contradicting the preliminary claims that MWT of wheat kernels detoxifies gluten. This study provides evidence that R5-antibody ELISA alone is not effective to determine gluten in thermally treated wheat products. Gluten epitopes in processed wheat should be monitored using strategies based on combined immunoassays with T cells from celiacs, G12-antibody ELISA after proteolysis and proper molecular characterization.

  8. Characterization of an immunodominant cancer-specific O-glycopeptide epitope in murine podoplanin (OTS8)

    DEFF Research Database (Denmark)

    Steentoft, Catharina; Schjoldager, Katrine T; Cló, Emiliano

    2010-01-01

    of the nature and timing of induction of auto-antibodies to distinct O-glycopeptide epitopes induced by cancer. The results demonstrate that truncated O-glycopeptides constitute highly distinct antibody epitopes with great potential as targets for biomarkers and immunotherapeutics.......Auto-antibodies induced by cancer represent promising sensitive biomarkers and probes to identify immunotherapeutic targets without immunological tolerance. Surprisingly few epitopes for such auto-antibodies have been identified to date. Recently, a cancer-specific syngeneic murine monoclonal...... antibody 237, developed to a spontaneous murine fibrosarcoma, was shown to be directed to murine podoplanin (OTS8) with truncated Tn O-glycans. Our understanding of such cancer-specific auto-antibodies to truncated glycoforms of glycoproteins is limited. Here we have investigated immunogenicity...

  9. NetCTLpan: pan-specific MHC class I pathway epitope predictions

    DEFF Research Database (Denmark)

    Stranzl, Thomas; Larsen, Mette Voldby; Lundegaard, Claus;

    2010-01-01

    molecules with known protein sequence and allows predictions for 8-, 9-, 10-, and 11-mer peptides. In order to meet the need for a low false positive rate, the method is optimized to achieve high specificity. The method was trained and validated on large datasets of experimentally identified MHC class I......Reliable predictions of immunogenic peptides are essential in rational vaccine design and can minimize the experimental effort needed to identify epitopes. In this work, we describe a pan-specific major histocompatibility complex (MHC) class I epitope predictor, NetCTLpan. The method integrates...... cleavage and TAP transport for all MHC molecules. The predictive performance of the NetCTLpan method was shown to outperform other state-of-the-art CTL epitope prediction methods. Our results further confirm the importance of using full-type human leukocyte antigen restriction information when identifying...

  10. Select human anthrax protective antigen (PA) epitope-specific antibodies provide protection from lethal toxin challenge

    Science.gov (United States)

    Crowe, Sherry R.; Ash, Linda L.; Engler, Renata J. M.; Ballard, Jimmy D.; Harley, John B.; Farris, A. Darise; James, Judith A.

    2010-01-01

    Bacillus anthracis remains a serious bioterrorism concern, and the currently licensed vaccine remains an incomplete solution for population protection from inhalation anthrax and has been associated with concerns regarding efficacy and safety. Thus, understanding how to generate long lasting protective immunity with reduced immunizations or providing protection through post exposure immunotherapeutics are long sought goals. Through evaluation of a large military cohort, we characterized the levels of antibodies against protective antigen and found that over half of anthrax vaccinees had low levels of in vitro toxin neutralization capacity in their sera. Using solid phase epitope mapping and confirmatory assays, we identified several neutralization-associated humoral epitopes and demonstrated that select anti-peptide responses mediated protection in vitro. Finally, passively transferred antibodies specific for select epitopes provided protection in an in vivo lethal toxin mouse model. Identification of these antigenic regions has important implications for vaccine design and the development of directed immunotherapeutics. PMID:20533877

  11. Recognition and sensing of low-epitope targets via ternary complexes with oligonucleotides and synthetic receptors

    Science.gov (United States)

    Yang, Kyung-Ae; Barbu, Mihaela; Halim, Marlin; Pallavi, Payal; Kim, Benjamin; Kolpashchikov, Dmitry M.; Pecic, Stevan; Taylor, Steven; Worgall, Tilla S.; Stojanovic, Milan N.

    2014-11-01

    Oligonucleotide-based receptors or aptamers can interact with small molecules, but the ability to achieve high-affinity and specificity of these interactions depends strongly on functional groups or epitopes displayed by the binding targets. Some classes of targets are particularly challenging: for example, monosaccharides have scarce functionalities and no aptamers have been reported to recognize, let alone distinguish from each other, glucose and other hexoses. Here we report aptamers that differentiate low-epitope targets such as glucose, fructose or galactose by forming ternary complexes with high-epitope organic receptors for monosaccharides. In a follow-up example, we expand this method to isolate high-affinity oligonucleotides against aromatic amino acids complexed in situ with a nonspecific organometallic receptor. The method is general and enables broad clinical use of aptamers for the detection of small molecules in mix-and-measure assays, as demonstrated by monitoring postprandial waves of phenylalanine in human subjects.

  12. Depigmented Allergoids Reveal New Epitopes with Capacity to Induce IgG Blocking Antibodies

    Directory of Open Access Journals (Sweden)

    M. Angeles López-Matas

    2013-01-01

    Full Text Available Background. The synthesis of allergen-specific blocking IgGs that interact with IgE after allergen immunotherapy (SIT has been related to clinical efficacy. The objectives were to investigate the epitope specificity of IgG-antibodies induced by depigmented-polymerized (Dpg-Pol allergoids and unmodified allergen extracts, and examine IgE-blocking activity of induced IgG-antibodies. Methods. Rabbits were immunized with native and Dpg-Pol extracts of birch pollen, and serum samples were obtained. Recognition of linear IgG-epitopes of Bet v 1 and Bet v 2 and the capacity of these IgG-antibodies to block binding of human-IgE was determined. Results. Serum from rabbits immunized with native extracts recognised 11 linear epitopes from Bet v 1, while that from Dpg-Pol-immunized animals recognised 8. For Bet v 2, 8 epitopes were recognized by IgG from native immunized animals, and 9 from Dpg-Pol immunized one. Dpg-Pol and native immunized serum did not always recognise the same epitopes, but specific-IgG from both could block human-IgE binding sites for native extract. Conclusions. Depigmented-polymerized birch extract stimulates the synthesis of specific IgG-antibodies which recognize common but also novel epitopes compared with native extracts. IgG-antibodies induced by Dpg-Pol effectively inhibit human-IgE binding to allergens which may be part of the mechanism of action of SIT.

  13. Identification of a major epitope recognized by PLA2R autoantibodies in primary membranous nephropathy.

    Science.gov (United States)

    Fresquet, Maryline; Jowitt, Thomas A; Gummadova, Jennet; Collins, Richard; O'Cualain, Ronan; McKenzie, Edward A; Lennon, Rachel; Brenchley, Paul E

    2015-02-01

    Phospholipase A2 receptor 1 (PLA2R) is a target autoantigen in 70% of patients with idiopathic membranous nephropathy. We describe the location of a major epitope in the N-terminal cysteine-rich ricin domain of PLA2R that is recognized by 90% of human anti-PLA2R autoantibodies. The epitope was sensitive to reduction and SDS denaturation in the isolated ricin domain and the larger fragment containing the ricin, fibronectin type II, first and second C-type lectin domains (CTLD). However, in nondenaturing conditions the epitope was protected against reduction in larger fragments, including the full-length extracellular region of PLA2R. To determine the composition of the epitope, we isolated immunoreactive tryptic fragments by Western blotting and analyzed them by mass spectrometry. The identified peptides were tested as inhibitors of autoantibody binding to PLA2R by surface plasmon resonance. Two peptides from the ricin domain showed strong inhibition, with a longer sequence covering both peptides (31-mer) producing 85% inhibition of autoantibody binding to PLA2R. Anti-PLA2R antibody directly bound this 31-mer peptide under nondenaturing conditions and binding was sensitive to reduction. Analysis of PLA2R and the PLA2R-anti-PLA2R complex using electron microscopy and homology-based representations allowed us to generate a structural model of this major epitope and its antibody binding site, which is independent of pH-induced conformational change in PLA2R. Identification of this major PLA2R epitope will enable further therapeutic advances for patients with idiopathic membranous nephropathy, including antibody inhibition therapy and immunoadsorption of circulating autoantibodies.

  14. Enlarging the toolbox for allergen epitope definition with an allergen-type model protein.

    Directory of Open Access Journals (Sweden)

    Hanna Berkner

    Full Text Available Birch pollen-allergic subjects produce polyclonal cross-reactive IgE antibodies that mediate pollen-associated food allergies. The major allergen Bet v 1 and its homologs in plant foods bind IgE in their native protein conformation. Information on location, number and clinical relevance of IgE epitopes is limited. We addressed the use of an allergen-related protein model to identify amino acids critical for IgE binding of PR-10 allergens.Norcoclaurine synthase (NCS from meadow rue is structurally homologous to Bet v 1 but does not bind Bet v 1-reactive IgE. NCS was used as the template for epitope grafting. NCS variants were tested with sera from 70 birch pollen allergic subjects and with monoclonal antibody BV16 reported to compete with IgE binding to Bet v 1.We generated an NCS variant (Δ29NCSN57/I58E/D60N/V63P/D68K harboring an IgE epitope of Bet v 1. Bet v 1-type protein folding of the NCS variant was evaluated by 1H-15N-HSQC NMR spectroscopy. BV16 bound the NCS variant and 71% (50/70 sera of our study population showed significant IgE binding. We observed IgE and BV16 cross-reactivity to the epitope presented by the NCS variant in a subgroup of Bet v 1-related allergens. Moreover BV16 blocked IgE binding to the NCS variant. Antibody cross-reactivity depended on a defined orientation of amino acids within the Bet v 1-type conformation.Our system allows the evaluation of patient-specific epitope profiles and will facilitate both the identification of clinically relevant epitopes as biomarkers and the monitoring of therapeutic outcomes to improve diagnosis, prognosis, and therapy of allergies caused by PR-10 proteins.

  15. Defining species-specific immunodominant B cell epitopes for molecular serology of Chlamydia species.

    Science.gov (United States)

    Rahman, K Shamsur; Chowdhury, Erfan U; Poudel, Anil; Ruettger, Anke; Sachse, Konrad; Kaltenboeck, Bernhard

    2015-05-01

    Urgently needed species-specific enzyme-linked immunosorbent assays (ELISAs) for the detection of antibodies against Chlamydia spp. have been elusive due to high cross-reactivity of chlamydial antigens. To identify Chlamydia species-specific B cell epitopes for such assays, we ranked the potential epitopes of immunodominant chlamydial proteins that are polymorphic among all Chlamydia species. High-scoring peptides were synthesized with N-terminal biotin, followed by a serine-glycine-serine-glycine spacer, immobilized onto streptavidin-coated microtiter plates, and tested with mono-specific mouse hyperimmune sera against each Chlamydia species in chemiluminescent ELISAs. For each of nine Chlamydia species, three to nine dominant polymorphic B cell epitope regions were identified on OmpA, CT618, PmpD, IncA, CT529, CT442, IncG, Omp2, TarP, and IncE proteins. Peptides corresponding to 16- to 40-amino-acid species-specific sequences of these epitopes reacted highly and with absolute specificity with homologous, but not heterologous, Chlamydia monospecies-specific sera. Host-independent reactivity of such epitopes was confirmed by testing of six C. pecorum-specific peptides from five proteins with C. pecorum-reactive sera from cattle, the natural host of C. pecorum. The probability of cross-reactivity of peptide antigens from closely related chlamydial species or strains correlated with percent sequence identity and declined to zero at Chlamydia spp. We anticipate that these peptide antigens will improve chlamydial serology by providing easily accessible assays to nonspecialist laboratories. Our approach also lends itself to the identification of relevant epitopes of other microbial pathogens.

  16. GPS-MBA: computational analysis of MHC class II epitopes in type 1 diabetes.

    Science.gov (United States)

    Cai, Ruikun; Liu, Zexian; Ren, Jian; Ma, Chuang; Gao, Tianshun; Zhou, Yanhong; Yang, Qing; Xue, Yu

    2012-01-01

    As a severe chronic metabolic disease and autoimmune disorder, type 1 diabetes (T1D) affects millions of people world-wide. Recent advances in antigen-based immunotherapy have provided a great opportunity for further treating T1D with a high degree of selectivity. It is reported that MHC class II I-A(g7) in the non-obese diabetic (NOD) mouse and human HLA-DQ8 are strongly linked to susceptibility to T1D. Thus, the identification of new I-A(g7) and HLA-DQ8 epitopes would be of great help to further experimental and biomedical manipulation efforts. In this study, a novel GPS-MBA (MHC Binding Analyzer) software package was developed for the prediction of I-A(g7) and HLA-DQ8 epitopes. Using experimentally identified epitopes as the training data sets, a previously developed GPS (Group-based Prediction System) algorithm was adopted and improved. By extensive evaluation and comparison, the GPS-MBA performance was found to be much better than other tools of this type. With this powerful tool, we predicted a number of potentially new I-A(g7) and HLA-DQ8 epitopes. Furthermore, we designed a T1D epitope database (TEDB) for all of the experimentally identified and predicted T1D-associated epitopes. Taken together, this computational prediction result and analysis provides a starting point for further experimental considerations, and GPS-MBA is demonstrated to be a useful tool for generating starting information for experimentalists. The GPS-MBA is freely accessible for academic researchers at: http://mba.biocuckoo.org.

  17. Selection of Conserved Epitopes from Hepatitis C Virus for Pan-Populational Stimulation of T-Cell Responses

    Science.gov (United States)

    Molero-Abraham, Magdalena; Lafuente, Esther M.; Flower, Darren R.; Reche, Pedro A.

    2013-01-01

    The hepatitis C virus (HCV) is able to persist as a chronic infection, which can lead to cirrhosis and liver cancer. There is evidence that clearance of HCV is linked to strong responses by CD8 cytotoxic T lymphocytes (CTLs), suggesting that eliciting CTL responses against HCV through an epitope-based vaccine could prove an effective means of immunization. However, HCV genomic plasticity as well as the polymorphisms of HLA I molecules restricting CD8 T-cell responses challenges the selection of epitopes for a widely protective vaccine. Here, we devised an approach to overcome these limitations. From available databases, we first collected a set of 245 HCV-specific CD8 T-cell epitopes, all known to be targeted in the course of a natural infection in humans. After a sequence variability analysis, we next identified 17 highly invariant epitopes. Subsequently, we predicted the epitope HLA I binding profiles that determine their potential presentation and recognition. Finally, using the relevant HLA I-genetic frequencies, we identified various epitope subsets encompassing 6 conserved HCV-specific CTL epitopes each predicted to elicit an effective T-cell response in any individual regardless of their HLA I background. We implemented this epitope selection approach for free public use at the EPISOPT web server. PMID:24348677

  18. In silico prediction of B cell epitopes of the extracellular domain of insulin-like growth factor-1 receptor

    Directory of Open Access Journals (Sweden)

    Vahid Bayrami

    2016-12-01

    Full Text Available The insulin-like growth factor-1 receptor (IGF-1R is a transmembrane receptor with tyrosine kinase activity. The receptor plays a critical role in cancer. Using monoclonal antibodies (MAbs against the IGF-1R, typically blocks ligand binding and enhances down-regulation of the cell-surface IGF-1R. Some MAbs such as cixutumumab are under clinical trial investigation. Targeting multiple distinct epitopes on IGF-1R, might be an effective strategy to inhibit IGF-1R pathway in cancer. In this study, new linear B cell epitopes for the extracellular domains of IGF-1R were predicted by in silico methods using a combination of linear B cell epitope prediction web servers such as ABCpred, Bepired, BCPREDs, Bcepred and Elliprro. Moreover, Discotope, B-pred and PEPOP web server tools were employed to predict new conformational B cell epitopes. In contrast to previously reported epitopes from extracellular region of the IGF-1R, we predicted new linear P8: (RQPQDGYLYRHNYCSK and conformational Pc4: (HYYYAGVCVPACPPNTYRFE, Ppc6: (KMCPSTGKRENNESAPDNDT and Ppc20: (ANILSAESSDSEFMQEPSGFI epitopes. These epitopes are useful for further study as peptide antigens to actively immune host animals to develop new MAbs. Furthermore, the epitopes can be used in peptide-based cancer vaccines design.

  19. Vaccine generated immunity targets an HPV16 E7 HLA-A2.1-restricted CD8(+) T cell epitope relocated to an early gene or a late gene of the cottontail rabbit papillomavirus (CRPV) genome in HLA-A2.1 transgenic rabbits.

    Science.gov (United States)

    Bounds, Callie E; Hu, Jiafen; Cladel, Nancy M; Balogh, Karla; Christensen, Neil D

    2011-02-01

    The newly established HLA-A2.1 transgenic rabbit model has proven useful for testing the immunogenicity of well known and computer-predicted A2-restricted epitopes. In the current study we compared the protective immunity induced to a preferred HPV16 E7 A2-restricted epitope that has been relocated to positions within the CRPV E7 gene and the CRPV L2 gene. Epitope expression from both the E7 protein and the L2 protein resulted in increased protection against viral DNA challenge of the HLA-A2.1 transgenic rabbits as compared to control-vaccinated rabbit groups. These data indicate that proteins expressed at both early and late time points during a natural papillomavirus infection can be targeted by epitope-specific immunity and indicate this immunity is increased to early rather than late expressed proteins of papillomaviruses. This study also highlights the broad utility of the HLAA2.1 transgenic rabbit model for testing numerous immunological factors involved in vaccine generated protective immunity.

  20. Interaction of an immunodominant epitope with Ia molecules in T-cell activation

    DEFF Research Database (Denmark)

    Adorini, L; Sette, A; Buus, S;

    1988-01-01

    The amino acid sequence corresponding to residues 107-116 of hen egg-white lysozyme (HEL) has been identified as containing an immunodominant T-cell epitope recognized in association with the I-Ed molecule. The immunodominance of this epitope in HEL-primed H-2d mice was demonstrated by analysis o......-120)-peptide was found to be immunogenic in H-2d mice. Thus, a single semiconservative substitution drastically reduces binding capacity and abolishes immunogenicity, suggesting that a strict correlation exists between binding of a peptide to Ia molecules and its immunogenicity....